THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed m U.S.A. Vol. 255. No. 3, Issue of February IO, pp. 1170-1174, 1980

Factor Va-dependent Binding of Factor X, to Human Platelets*

(Received for publication, May 15, 1979)

William H. Kane, M. J. Lindhout, Craig M. Jackson, and Philip W. Majerus From the Division of Hematology-Oncology, Departments of Internal Medicine and Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110

Coagulation Factor V from platelets has a role in platelet-Factor X, binding and prothrombin activation. We now find that when human platelets are stimulated to undergo the release reaction with thrombin they release 0.170 to 0.350 unit of Factor V (V,) activity/108 platelets. The activity reaches a maximum 5 to 10 min after thrombin addition and is not further increased by subsequent treatment with thrombin, suggesting that it is V,. [‘4C]Serotonin release is complete within 30 s and the thrombin concentration required for [14C]ser- otonin and Factor V activity release are the same, with half-maximal [14C]serotonin release at 0.02 unit of thrombin/ml and half-maximal Factor V activity re- lease at 0.015 unit of thrombin/ml. When arachidonic acid or calcium ionophore A23187 are used to induce the platelet release reaction, Factor V is released as the procofactor. Thus, the Factor V activity released by these agents can be activated from 10- to 20-fold with either thrombin or the Factor V-activating protein from Russell’s viper venom (V-CP). This activation of plate- let Factor V following release is similar to that obtained using purified bovine Factor V.

We used the thrombin inhibitor dansyl-arginine 4- ethyl piperidine amide to block thrombin stimulation of the platelet release reaction during platelet X,-bind- ing studies. Platelets which are protected from throm- bin stimulation by dansyl-arginine 4-ethyl piperidine amide do not bind human Factor X, either in the pres- ence or absence of bovine Factor V. However, unstim- ulated platelets incubated with bovine Factor V, (1 pg/ ml) bind Factor X, (2.0 ng of ‘251-Fa~tor X,/108, Kd 70 PM) with the same affinity and number of binding sites as thrombin-stimulated platelets. We conclude that Factor V activation is necessary for platelet Factor X, binding and that the “sites” which bind Factor X, are on the surface of unstimulated platelets.

We have outlined the role of platelets in prothrombin activation (1-3).’ After stimulation of the release reaction, 200 to 300 Factor X, molecules bind per platelet with an apparent dissociation constant of 30 PM. Platelet Factor X, binding sites are specific for X,: prothrombin, Factor X, Factor IX, or Factor IX, do not bind. Factor X, bound to platelets catalyzes the activation of prothrombin 300,000 times faster than Factor

* This research was supported by Grants HLBI 14147 (Specialized Center in Thrombosis), HL 12820, and HL 16634 from the National Institutes of Health, and in part by National Institutes of Health Research Service Award GM 07200, Medical Scientist, from the National Institute of General Medical Sciences. The costs of publi- cation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I J. P. Miletich, W. H. Kane, S. L. Hofmann, N. Stanford, and P. W. Majerus (1979) Blood 54, 1015-1022.

X. in solution. Factor V is required for the binding of Factor X. to platelets since either normal platelets treated with an antibody to Factor V or platelets from Factor V-deficient individuals (3) show decreased Factor X, binding and Factor X,-catalyzed prothrombin activation. The addition of purified bovine Factor V. o r the Factor V (actually Factor V,, see below) activity released by thrombin from normal human platelets corrects Factor X, binding to Factor V-deficient platelets, but does not increase Factor X, binding to control platelets. Because the small number of Factor X, binding sites could not be increased by Factor V, we concluded that some component other than Factor V, limited the number of bind- ing sites. We recently described a patient with a moderately severe bleeding disorder who had normal plasma coagulation factors and platelet Factor V,’ but abnormal platelet Factor X, binding and Factor X,-catalyzed prothrombin activation. This patient’s platelets are deficient in the platelet component to which factors X, and V. bind. These results and the correlation between Factor X, binding and severity of bleeding in Factor V-deficient patients indicate that platelet surface prothrombin activation is important in normal hemostasis.

Factor V has recently been purified to homogeneity from bovine plasma and its proteolytic activation by thrombin characterized (4-6). These results indicate that Factor V is a minimally active procofactor composed of a single polypeptide chain of M , 330,000 which is hydrolyzed by thrombin to the active form V,. Previous studies indicated that activation was necessary for binding of Factor V, to prothrombin (7) and Factor X, (8).

We now report that Factor V released from human platelets in the absence of thrombin can be activated to the same extent as our purified bovine Factor V preparation. Bovine Factor V, but not bovine Factor V allows unstimulated platelets to bind Factor X, in the same manner as Factor X. binds to platelets that have undergone the release reaction as a result of thrombin stimulation. Therefore, we conclude that the sites to which Factor X, binds are present on the surface of unstim- ulated platelets and that the previously observed requirement for platelet stimulation reflects the requirement for Factor V release and activation since the platelets were the source of Factor V in those experiments.

MATERIALS AND METHODS

The preparation and assay of human prothrombin, thrombin, and factor X. has been described (2). Bovine Factor V was prepared by a modification‘of the method of Esmon (5.9). The Factor V preparation was stored at -70°C in a buffer containing 0.5 M NHdCl, 50 mM Tris- HCI, pH 7.4, and 10 mM benzamidine. Prior to use, the benzamidine was removed by passing the preparation over a 1-ml column of Sephadex G-25 equilibrated with 0.15 M NaCl, 20 mM Tris, pH 7.4. Protein concentration was estimated from absorbance at 280 nm assuming E& = 9.6 (4). Bovine V (0.2 to 0.4 mg/ml) was converted to V, by a 15-min incubation at 37°C with either 1 unit/ml of thrombin or 10 pg/ml of the Factor V-activating enzyme from Rus-

M. J. Lindhout and C. M. Jackson, manuscript in preparation.

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Factor X , Binding Sites 4

sell's viper venom (V-CP)' which was purified by the method of Esmon (9). Sodium dodecyl sulfate polyacrylamide gel electrophoresis was done as described by Laemmli (10) using 10% acrylamide with a 4% acrylamide stacking gel. Molecular weight standards (Bio-Rad) were phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, and lysozyme.

Bovine Factor V and platelet Factor V activity were assayed in a one-stage assay system (3) with a functional range from 0.3 to 30 milliunits/ml. The amount of V activity in citrated human plasma was defined IF, 1 unit/ml.

Methods for iodinating Factor X. and measuring human platelet I2'II-Factor X, binding and ['4C]serotonin release have been described (1, 2). Factor X. bound was corrected for nonspecific binding by subtracting the amount of Factor "'I-X bound in parallel reaction mixtu..,s containing 1 pg of unlabeled Factor X/ml. Nonspecific binding was 2 to 5% of total X. added. In some experiments Iz5I-

Factor X. binding and ['4C]serotonin release were determined simul- taneously. [I4C]Serotonin was determined by liquid scintillation counting after correcting for radioactivity.

Dansyl-arginine-4-ethyl piperidine amide (DAPA) was a gift of Doctors Michael Nesheim and Kenneth Mann (Mayo Clinic, Roch- ester, MN).

Bovine Factor V Preparation-Since pure human Factor V is not available, bovine Factor V was used in our studies. The Factor V preparation gave one high molecular weight band upon electropho- resis in polyacrylamide gels with sodium dodecyl sulfate and 2-mer- captoethanol (Fig. 1). Thrombin-activated V. showed two major Coomassie blue-staining bands. One band with apparent M, = 94,000 apparently corresponds with the M, = 115,000 Factor V. heavy chain of Esmon (5) and the M, = 94,000 activation fragment of Nesheim and Mann (6). The other band with apparent M, = 72,000 corresponds to the Factor V. light chain of Esmon and to either the Mr = 74,000 or 72,000 activation fragment of Nesheim and Mann. The remainder of the mass of the Factor V molecule (M, = 330,000) is presumably accounted for by the several other poorly staining bands present which have yet to be characterized. Factor V in plasma is a procofactor that can be converted to the active species V. by either thrombin or V-CP (11-15). Although the cleavage pattern obtained with V-CP is different from that obtained with thrombin, the specific activity as measured by coagulation assay is the same (14, 15).' The apparent activity of the procofactor V, as measured by coagulation assay, is probably due in part to small amounts of V. which are formed as some of the procofactor is converted to V. during the assay. Our bovine Factor V preparation had a specific activity of 140 units/mg; after activation by thrombin the specific activity was 2000 units/mg. This 14-fold increase in specific activity is the same as that which was observed when Factor V in bovine plasma was activated by V-CP, as measured by our assay.

RESULTS

Release of Factor V Activity from Platelets-When plate- lets were stimulated to undergo the release with 0.5 unit/ml thrombin (Fig. 2), [I4C]serotonin release reached maximal levels in less than 30 s. Factor V (actually Factor V., see below) activity appeared more slowly in platelet supernatants, reaching a maximum after 10 to 15 min, with 50% released in 2 min. All samples were exposed to 0.5 unit/ml of thrombin for at least 20 min before being assayed. Twenty minutes is sufficient for complete profactor activation (data not shown); thus the measured increase in Factor V activity was due solely to release of Factor V and not to profactor activation. In six experiments, the amount of Factor V. activity released ranged from 0.170 to 0.350 unit/lO" platelets. Factor V. activity in platelet supernatants was stable in the presence of 5 mM CaCI2 for a t least 90 min. However, when supernatants were diluted 1:500 for assay, the Factor V. activity was unstable, declining to less than 10% of the original activity within 10 min. Con- sequently, samples were assayed immediately after dilution. Lability of Factor V activity was not observed following dilu-

The abbreviations used are: "%Factor X., '2511-labeled Factor X.; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; DAPA, dansyl-argi- nine-4-ethyl piperidine amide; V-CP, Factor V activating protein from Russell's viper venom.

3n Unstimulated Platelets 1171

FIG. 1. Sodium dodecyl sulfate-gel electrophoresis of bovine Factor V and thrombin-activated bovine V. in 10% polyacryl- amide with a 4% polyacrylamide stacking gel, stained with Coomassie brilliant blue. Left, 5 pg of reduced bovine Factor V. Right, 5 pg of reduced thrombin-activated bovine V.. Arrows indicate top of 10% acrylamide gel.

tion of purified bovine Factor V or samples of human plasma. The thrombin concentrations required for 50% [I4C]serotonin release (0.02 unit/ml) and 50% Factor V. activity release (0.015 unit/ml) were similar (Fig. 3). The Factor V activity measured in this experiment represents fully activated Factor V. since it was not further activated by treatment with thrombin (data not shown). The Factor V. activity released by thrombin treatment was equal to the total Factor V activity measured after platelets were frozen and thawed three times.

Activation of Platelet Factor V-Supernatants from un- stimulated, washed platelets contained small amounts of Fac- tor V activity (<0.0005 unit/lO" platelets). Platelets incubated with 0.5 unit/ml of thrombin for 10 min released Factor V activity that was not further activated by treatment with 10 pg/ml of V-CP, presumably because it was already fully activated to Factor V. by the thrombin used to induce the release reaction. Platelets are more sensitive to stimulation by thrombin in buffers of low ionic strength because of enhanced thrombin binding (16). In an effort to separate the effect of thrombin in stimulating the release reaction from its effect on Factor V, we treated platelets with 0.005 unit/ml of thrombin in a buffer containing 34 mM NaCI, 20 m~ Tris, pH 7.4, 0.21 M glucose, and 5 mg/ml of bovine serum albumin (conductivity 3.08 mmho). In this experiment we found 0.004 unit of Factor V/lO" platelets released after 10 min. This activity was acti- vated 5-fold upon subsequent incubation with 0.25 u n i t / d of

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1172 Factor X, Binding Sites on Unstimulated Platelets 0

5 IO 15 20 MINUTES

FIG. 2. Time course of the release of platelet Factor V activ-

treated platelets. Platelets (10'/ml) in 0.15 M NaCl, 20 mM Tris- ity and [''C]serotonin into the supernatant of thrombin-

HC1, 5.5 mM glucose, 5 mM CaC12, 5 mg/ml of bovine serum albumin, pH 7.4, were incubated with 0.5 unit/ml of thrombin at room tem- perature. Samples were taken and centrifuged through oil at the indicated times. After the last sample was collected, the platelet supernatants were assayed for Factor V activity in random order, 0. ['4C]Serotonin was measured in the platelet supernatants and per cent serotonin release was determined, 0. In this experiment, the maximum Factor V activity released was 0.25 unit/lOH platelets; maximum ['4C]serotonin release was 89%.

1 0.01

THROMBIN (U/ml) 0.1 1.0

FIG. 3. Thrombin dose-response curve for platelet Factor V activity and ['4C]serotonin release from human platelets. Plate- let Factor V, 0, activity and [14C]serotonin, 0, were measured as in Fig. 2. Platelets were incubated with different amounts of thrombin for 20 min. The platelet suspension were then centrifuged through oil, and platelet supernatants assayed in random order. In this exper- iment, the maximum Factor V activity released was 0.24 unitll0" platelets, maximum ["Clserotonin release was 82%.

thrombin to 0.021 unit of Factor V/108 platelets. Finally, platelets stimulated with 0.5 p~ arachidonic acid in a buffer containing 0.15 M NaCl, 20 mhf Tris, pH 7.4, 5.5 mM glucose, released a small amount of Factor V activity, after a 10-min incubation, which subsequently increased 10- to 25-fold upon incubation with V-CP (z.e. 0.0013 to 0.031 unit of Factor V/ 10" platelets). Similar results were obtained in experiments using 0.5 IJM calcium ionophore A23187, namely that small amounts of Factor V activity were released from platelets which was subsequently activated by either thrombin or V- CP (i.e. from 0.008 to 0.089 unit of Factor V/108 platelets). These results suggest that platelet Factor V is released pri- marily as a procofactor and that Factor V is not rapidly activated after the release reaction in the absence of thrombin. Since the coagulation assay is only a semiquantitative assess- ment of the presence of Factor V uersus Factor V, we cannot exclude partial activation of Factor V released from platelets.

Binding of '251-labeled Factor X , to Unstimulated Plate- lets-We previously concluded that the platelet release reac- tion was required to make Factor X, receptor sites available (1, 2). However, since our previous experiments (1, 2) were carried out in the presence of prothrombin and Factor X,, thrombin was generated, thereby obscuring the distinction between Factor V release and Factor V activation. We thus pose two questions: 1) Are the binding sites for factor X, present on unstimulated platelets or is the release reaction required for their expression? 2) Will Factor V allow platelets to bind X, or is Factor V, required? In order to study the binding of '251-Fa~tor X, to unstimulated platelets in the presence of prothrombin, we used an inhibitor of thrombin, DAPA. DAPA is a nonhydrolyzable competitive inhibitor of thrombin with a K, of 5 X M for synthetic substrates (17). It does not affect thrombin formation from prothrombin by Factor X, and Factor V,. It has previously been described as

When platelets were incubated with '251-Fa~tor X,, 5 mM CaC12, 75 pg/ml of prothrombin, and 93 IJM DAPA, no Factor X, binding was observed (Fig. 4). Platelets did not undergo the release reaction during the binding assay since [I4C]sero- tonin release was 4 0 % . This c o n f m s previous results where no Factor X, binding to unstimulated platelets was observed when prothrombin was omitted from the binding assay (1,19). When the above experiment was done in the presence of 1 pg/ ml of V-CP-activated bovine V,, Factor X, binding to platelets was observed despite the fact that the platelets had not undergone the release reaction as measured by [I4C]serotonin release. When data from this experiment are plotted as a double reciprocal plot (not shown), the maximum amount of specific X, binding was 2.0 ng/lOH platelets with an apparent dissociation constant of 70 PM, values similar to those obtained previously in studies with thrombin-treated platelets (1.65 f 0.4 ng of Factor X,/lOx platelets and K,I = 30 PM) (1-3). When thrombin-activated bovine V, was used, similar results were

OM-205 (18).

.-- - 1 ' 5 10 15 20

a ..,I

Xa FREE ( n g / m l l

FIG. 4. 12'I-Factor X. binding of human platelets. Platelets (lO'/ml) were incubated with the "'I-Factor X. in a mixture contain- ing 0.15 M NaCI, 20 mM Tris-HC1, 5.5 mM glucose, 5 m g / d of bovine serum albumin, 5 mM CaCI2, 75 pg/ml of prothrombin for 30 min. A, A, plus 93 p~ DAPA; B, 0, plus 1 p g / d of V-CP-activated bovine V, and 93 PM DAPA; C, 0, platelets were stimulated with 0.1 unit/ml of thrombin; 0, 0, platelets were stimulated with 0.1 unit/ml of throm- bin, 93 PM DAPA was added 10 min later. The specific radioactivity of the Factor X. preparation used in this experiment was 1300 cpm/ ng. Data were corrected for nonspecific binding which was 4% of the radioactivity added.

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Factor X , Binding Sites on Unstimulated Platelets 1173

BOVINE V or Va ( n Q / m l )

FIG. 5. 12'I-Factor X. binding to unstimulated platelets in the presence of bovine V or bovine V.. The binding of "'I-Factor X. to unstimulated platelets was determined as in Fig. 4B in the presence of 7 ng/ml of '"I-Factor X. and varying concentrations of thrombin-activated bovine V. 0, or bovine V, 0. Platelets stimulated with 0.1 unit/ml of thrombin and incubated with 7 ng/ml of '"I- Factor X, with 93 p~ DAPA added after 10 min of incubation as in Fig. 4 0 bound 1.6 ng of L251-Fa~tor X,/108 platelets, *. The specific radioactivity of the Iz5I-Factor X. preparation used in this experiment was 2600 cpm/ng. Data were corrected for nonspecific binding which was 2% of the radioactivity added.

obtained, indicating that either thrombin or V-CP-activated bovine Factor V, are equally capable of interacting with Factor X, and unstimulated platelets. Platelets from the same donor treated with 0.1 unit/ml of thrombin in order to induce the release reaction, and then incubated with 5 mM CaC12 and 75 pg/ml of prothrombin, bound a maximum of 2.1 ng of Iz5I- Factor X,/lOH platelets with an apparent dissociation constant of 66 PM. These platelets released 90% of ['4C]serotonin. Platelets were also stimulated to undergo the release reaction with 0.1 unit/ml thrombin and incubated for 10 min in the presence of '2511-Factor X., 75 pg/ml of prothrombin and 5 mM CaC12 before addition of 93 p~ DAPA for the remainder of the incubation. In this case similar platelet Factor X, binding was observed, a maximum of 2.85 ng of '"I-Factor X,/lOH platelets bound with an apparent dissociation constant of 96 PM. The 40 to 50% enhancement of Factor X, binding to platelets that were stimulated with thrombin followed by DAPA addition, compared to Factor X, bound to thrombin- treated platelets without added DAPA, was observed consist- ently. It is possible that DAPA prevents thrombin proteolysis of either Factor V, or some other component of the system. In the experiment in Fig. 4 platelets incubated with DAPA plus 1 pg of bovine Factor X,/ml bound less Factor X, than the thrombin-stimulated platelets plus DAPA because 1 pg of bovine V, is not a saturating amount as seen in Fig. 5. Other experiments demonstrated that unstimulated platelets bind as much Factor X. as thrombin-stimulated platelets in the presence of DAPA when saturating concentrations of bovine Factor V, are used.

Binding of ""I-Factor X , to Unstimulated Platelets in the Presence of Bovine Factor V, or Bovine Factor V-When

I-Factor X, binding to platelets was examined as a function of bovine Factor V, concentration (Fig. 5), we found that half- maximal Factor X, binding occurred at approximately 400 ng of Factor VJml and that 1.7 ng of '"I-Factor X,/lOH platelets bound a t 3.2 pg of added Factor V,/ml. In this experiment thrombin-stimulated platelets with 93 p~ DAPA added after 10 min of incubation bound 1.6 ng of "'I-Factor X,/lOx plate- l e t ~ . ~ No '2sI-Factor X, binding to platelets was observed when bovine V was added to unstimulated platelets in the presence

The total "'I-X. concentration in these experiments was 7 ng/ml. These results are consistent with those shown in Fig. 4 where throm- bin stimulates platelets with 93 mM DAPA added after 10 min bound 1.8 ng of '"I-Factor X,,/lO' platelets at the free "%Factor X,, concen- tration of 7 ng/ml.

115

of 93 p~ DAPA. In another experiment, bovine Factor V up to 6.4 pg/ml did not inhibit '251-Factor X, binding to unstim- ulated platelets in the presence of 1.6 pg/ml of bovine Factor V,. This result suggests that Factor V does not compete with Factor V, in enhancing Factor X, binding in the presence of prothrombin. We cannot exclude the possibility that Factor V binds to platelets under some conditions; however, it does not seem to participate in a complex with platelet surface pro- thrombin and factor X,.

DISCUSSION

We previously reported that Factor X, binding sites appear on the surface of platelets only after the platelet release reaction (1). Later we found that Factor V, derived from platelets in our experiments, was required for the Factor X,- platelet interaction. Thus, it was not clear whether the platelet component to which Factors V, and X. bind was also ex- pressed only after the release reaction. By analogy to study of prothrombin with phospholipids in place of platelets, it seems likely that Factor V. accelerates platelet surface prothrombin activation by binding with Factor X, to platelet sites. Since the interaction between platelets and the proteins required for prothrombin activation leads to rapid thrombin formation, it was necessary to block thrombin action to study the binding of Factor X, to unstimulated platelets. Our current results using DAPA indicate that the sites upon which prothrombin activation occurs are present on the surface of unstimulated platelets. We find the same number of Factor X, binding sites with equal affinity for Factor X, on both unstimulated plate- lets and platelets that have undergone the release reaction. Prothrombin activation has been previously postulated to occur on negatively charged phospholipid surfaces. The neg- atively charged phospholipids on eukaryotic cells are thought to be confined to the inner leaflet of cell membranes (20). Whether the 200 to 300 platelet surface prothrombin activa- tion sites are of some specialized phospholipid structures on the outer surface of platelets remains to be elucidated. The finding of a patient deficient in these sites' suggests that some protein component is probably required. The physiological significance of the exposure of prothrombin activation sites on the surface of unstimulated platelets is obscure. I t is conceiv- able that these sites allow for generation of the small amounts of thrombin found early in hemostasis prior to the platelet release reaction (21).

Our studies also indicate that Factor V, rather than Factor V is required for Factor X, binding to platelets. Similar requirements for the activated form of Factor V were obtained in previous studies in that Factor V, binds to Factor X,- agarose while Factor V does not (8) and only Factor V, binds to prothrombin (7). We used bovine Factor V in our studies since human Factor V has not yet been isolated. We find = 0.25 unit of Factor V, is released/lOH platelets. When 0.1 X 10' normal platelets and 0.9 X 10' platelets from a patient with Factor V deficiency were mixed in 1 ml and treated with 0.5 unit of thrombin/ml, we found that 0.68 ng of X, bound to the platelets.' This value was approximately one-half the amount that bound to loH normal platelets (1.2 ng of X,/lOH platelets). Since 0.1 X loM normal platelets release approximately 0.025 unit of Factor V, activity, we estimate that this concentration of factor V is sufficient to allow half-maximal Factor X, binding. In contrast, 0.4 pg/ml (0.8 unit/ml) of bovine Factor V, was required to achieve half-maximal human Factor X, binding to human platelets. The difference could be due to differences between human and bovine Factor V,. We previ- ously showed that human and bovine thrombin differ 3- to 4- fold in their affinity for the human platelet thrombin receptor (22). Thus, it is possible that the factor V coagulation assay is

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1174 Factor X , Binding Sites on Unstimulated Platelets

not a good measure of that part of the Factor V, molecule that binds to the platelet surface sites.

Mann et al. (23) described Factor V activity associated with platelets in 1947. Breederveld (24) showed that Factor V activity was expressed after repeated freezing and thawing, and concluded that the localization of Factor V was within platelets. Recently, Chesney et al. (25) have shown that Factor V is localized in the granule fraction of platelets and is released by collagen. Release of Factor V induced by collagen is in- hibited by 2-deoxyglucose or antimycin A, suggesting that Factor V release is a secretory process. Bsterud et al. (26) have proposed that platelets contain a Factor V activator and an activated Factor V molecule. They showed that Factor V from frozen and thawed platelets could not be further acti- vated by thrombin, and that a supernatant from sonicated platelets could partially activate purified plasma Factor V. Since platelets contain several proteases (27-29), it is possible that Factor V is activated in frozen and thawed platelets by a platelet protease(s) that does not activate Factor V after the release reaction. We find that Factor V activity released from platelets treated with arachidonic acid or calcium ionophore A23187 has an activation quotient similar to that of procofac- tor bovine Factor V. Factor V activity released from platelets treated with 0.02 unit/ml of thrombin can be activated only 2-fold 5 min after stimulation and is fully activated in 20 min at 22°C (data not shown). Ittyerah et al. (30) found that Factor V activity released by collagen in bovine platelets had an activation quotient of 7.4, while their purified bovine Factor V preparation had an activation quotient of 18. When bovine platelet Factor V was precipitated using an anti-bovine Factor V antibody and analyzed by sodium dodecyl sulfate electro- phoresis, a single band at M , = 280,000 was seen. These results suggest that collagen also releases procofactor V from bovine platelets. Thus, it appears that Factor V activity is released from platelets mostly in the procofactor form and that, when thrombin is used to stimulate platelets to undergo the release reaction, conversion of the released Factor V to Factor V, occurs.

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W H Kane, M J Lindhout, C M Jackson and P W MajerusFactor Va-dependent binding of factor Xa to human platelets.

1980, 255:1170-1174.J. Biol. Chem. 

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