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Background: The biological variability of von Willebrand factor and the variability in assays can make diagnosis and subclassification of von Willebrand disease (VWD) difficult. We describe a case series of four patients with a typical history of VWD and prolonged closure time in the platelet function analyser (PFA-100) but initially a normal ratio of ristocetin cofactor activity (VWF:RCo) to von Willebrand factor antigen levels (VWF:Ag) for whom further diagnostics verified VWD type 2A., Methods: For the initial VWD diagnostics we measured VWF:Ag, VWF:RCo, platelet aggregation induced by ADP, ristocetin and collagen, closure time in the PFA-100 test, and platelet count. We used VWF multimer analysis and collagen binding capacity for extended diagnostics. VWD diagnostics were carried out as part of extensive laboratory screening to exclude other haemostatic defects., Results: Multimer analysis revealed the absence of ultralarge multimers in all 4 patients. Ristocetin-induced platelet aggregation was consistently diminished in three patients with hereditary VWD 2A but not in a patient with essential thrombocythaemia. After repeat testing, diminished VWF:RCo and collagen binding capacity (VWF:CB) could be identified in all patients. However, all four cases would have been missed if the initial VWD assays had been performed only once., Conclusions: A single measurement of a normal ratio of VWF:RCo/VWF:Ag does not exclude VWD 2A in patients with a typical history of VWD. The PFA-100 is suitable for screening. To ensure that no cases of VWD are missed, multimer analysis and repeat functional testing of platelet aggregation, VWF:RCo, and VWF:CB are necessary.

Background: Diagnosis of acquired von Willebrand syndrome (AVWS) remains challenging. Diagnostic algorithms suggest the use of factor VIII (FVIII:C), von Willebrand factor antigen (VWF:Ag), ristocetin cofactor (VWF:RCo), and collagen-binding capacity (VWF:CB), but the sensitivity of these and other laboratory tests for the diagnosis of AVWS is unknown., Objectives: To analyze the capacity of laboratory tests, including point-of-care testing (POCT), for the identification of patients with AVWS., Patients/methods: Thirty-five consecutive patients were enrolled with AVWS diagnosed because of a history of recent onset of bleeding, a negative family history of von Willebrand disease, and abnormal plasma VWF multimers., Results: According to our inclusion criteria, all patients had bleeding symptoms, and the VWF high molecular weight multimers were either decreased or absent. Regarding POCT, PFA-100 was inconclusive, due to anemia or thrombocytopenia, in 29%; the sensitivity was 80% in the remaining patients. The sensitivity of VWF:Ag (23%), VWF:RCo/Ag ratio < 0.7 (26%), VWF:CB/Ag ratio < 0.7 (46%), anti-VWF antibodies (15%) and VWF propeptide/Ag ratio (22%) was too low to rule out the disease. A combination of VWF:Ag < 50 IU dL(-1), VWF:RCo/Ag ratio < 0.7 and VWF:CB/Ag ratio < 0.8 yielded a sensitivity of 86%. Patients diagnosed only because of abnormal VWF multimers showed similar clinical characteristics as other patients., Conclusions: Early diagnosis of AVWS is difficult, due to lack of sensitivity of the tests used. A substantial number of patients present with normal or increased test results, emphasizing the importance of multimer analysis in all patients with suspected AVWS.

Von Willebrand disease is characterised by a quantitative (type 1) or qualitative (type 2) decrease in von Willebrand factor (vWF) a multimeric glycoprotein involved in primary haemostasis. The propeptide of von Willebrand, also named vWF antigen II (vWF:AgII), is released from platelets and endothelial cells and circulates in plasma as a glycoprotein of 100 kD. In the present study, we attempted to determine whether vWF:AgII level may provide information on the synthesis of vWF, specially in patients with von Willebrand disease (vWD). To elucidate that point, we developed an ELISA and quantify the vWF:AgII in normal individuals and in various vWD patients. The propeptide molar concentration was found to be 5 nM as compared to 31 nM for mature vWF. In normal individuals, the level of vWF:AgII was significantly decreased in females from O and A blood groups. In type 2 vWD patients the level of plasma vWF:AgII appears normal in the patients with normal level of platelet vWF. In type 2 B vWD characterised by increased affinity of mature vWF for platelet glycoprotein Ib, the vWF:AgII in contrast to the vWF antigen (vWF:Ag) was not decreased. In type 2A vWD patients the level of vWF:AgII was decreased in patients with absence of high molecular weight vWF in platelets and plasma but normal in patients with increased sensitivity to proteolysis. Finally, in type 1 vWD, some studied patients have a parallel decrease in vWF:AgII and vWF:Ag whereas in others, the vWF:Ag levels were much more affected than corresponding vWF:AgII levels, as observed in some type 2 vWD patients. Thus, in contrast to that already described, the plasma vWF:AgII level cannot discriminate type 1 from type 2 vWD patients. We conclude that the vWF:AgII measurement provides additional information on the mechanisms responsible for vWD and might also contribute to the classification of vWD patients.

Monoclonal antibodies (MoAbs 833 and D4H1) directed against human factor VIII (FVIII) have been produced on a large scale to measure VIII:CAg by two-site ELISA (Asserachrom VIII:CAg, Diagnostica Stago). F(ab')2 from MoAb 833 were used for coating and bound VIII:CAg was revealed with MoAb D4H1 coupled to peroxidase. Control plasma (100 VIII:CAg U dL-1 by comparing with the International Standard) was used as reference. The assay sensitivity was 0.1 U dL-1 VIII:CAg. No apparent effect of the plasma proteins was observed provided plasma dilution was > or = 5. Thus this ELISA allowed us to estimate VIII:CAg levels of 0.5 U dL-1 in plasma. Levels of VIII:CAg were similar to those of VIII:C (correlation coefficient r = 0.87) in plasma from normal individuals (32 cases) and in patients with von Willebrand disease of various types (30 cases). Among 294 patients with haemophilia A (HA), 161 had severe HA (VIII:C < 1 U dL-1). Among those patients, 124 were cross-reacting material (CRM) negative with undetectable VIII:CAg and 37 were CRM+ (VIII:CAg 1-31 U dL-1). In 42 patients with moderate HA (VIII:C 1-5 U dL-1), 33 were CRM reduced (VIII:CAg 0.5-8 U dL-1) and nine were CRM+ with a VIII:CAg/VIII:C ratio of 6-91 (mean 34.3). In mild HA (91 cases with VIII:C > or = 6 U dL-1), 29 patients were classified as CRM+ (VIII:C 6-57 U dL-1, VIII:CAg 17-130 U dL-1 and VIII:CAg/VIII:C ratio 1.8-13.7 (mean 4.51)). In 62 CRM reduced patients there was a linear correlation between VIII:C (6-39 U dL-1) and VIII:CAg (2-36 U dL-1) levels (r = 0.88). In conclusion, this sensitive assay allows us to distinguish the quantitative CRM reduced and negative from the qualitative (CRM+) abnormalities in haemophilia A.

A family is described in which 5 out of 8 children had a marked bleeding disorder. The children showed prolonged bleeding times, abnormal platelet retention upon passage of blood through a glass bead column, the Willebrand factor activity as measured by ristocetin in a washed platelet system was low. Factor VIII/von Willebrand factor protein levels were normal even so the factor VIII-procoagulant activity. Even the parents and one child without any bleeding tendency and normal bleeding times had a reduced Willebrand factor activity. In all these patients evidence of an abnormal protein was observed on crossed antigen-antibody electrophoresis indicating a qualitative defect of the factor VIII/von Willebrand factor protein.

Precipitation pattern of factor VIII related antigen (VIIIR:AG) with concanavalin A (Con A), a glycoprotein binding lectin, was investigated in normal subjects and patients with a variant of von Willebrand's disease (VWD) and disseminated intravascular coagulation (DIC). Decreased precipitation with Con A was demonstrated in patients with a variant of VWD in which VIIIR:AG showed an increase in electrophoretic mobility on crossed immunoelectrophoresis (CIE). Similar findings were obtained in patients with DIC which revealed normal electrophoretic mobility of VIIIR:AG. This procedure was assumed to be useful for the detection of a qualitative abnormality of the factor VIII protein.

Collagen cofactor (CCo), an activity of von Willebrand factor (vWF) which increases the rate of adhesion of human fixed washed platelets (FWP) to collagen, was measured in plasma from normal individuals and individuals with von Willebrand's disease (vWD). CCo in vWD plasma was compared to vWF antigen (vWF:Ag), ristocetin cofactor (RCo), factor VIII (VIII) coagulant activity (VIII:C) and the quantitative bleeding time. There was close correlation between CCo and VIII:C (r = 0.909), vWF:Ag (r = 0.975), and RCo (r = 0.936). However, there was no correlation between CCo and the quantitative bleeding time. Plasma CCo in type IIA vWD was markedly lower than vWF:Ag and the ratio of CCo/vWF:Ag was 0.08, which was less than a mean value of 0.92 in type I vWD. CCo activity in normal plasma was completely inhibited by monoclonal antibody CLB-RAg 201, an antibody that inhibits the binding of vWF to collagen, suggesting that the binding of vWF to collagen is required for the expression of CCo. Furthermore, the partial inhibition of CCo by monoclonal antibody CLB-RAg 35 that inhibits the binding of vWF to platelet in the presence of ristocetin, suggests that CCo is partly mediated through platelet membrane glycoprotein Ib. Large multimers of vWF:Ag in normal plasma were preferentially absorbed by collagen. These studies demonstrate that CCo is another functional activity of vWF and the measurement of CCo may be useful for the detection of new variant forms of vWD.

A method has been developed for the immunological quantitation of factor VIII:C Ag using a medium titre (50 new Oxford units) factor VIII:C antibody arising in a severe multitransfused haemophiliac. The method, which utilizes clotting inhibition in an agarose gel medium, gave close and significant correlation with the two-stage factor VIII:C procoagulant assay (r=0.83, P less than 0.01) for 54 normal subjects. Similar or higher values were found in 19 mild, moderate and severe haemophiliacs with 2-30% average normal plasma levels of factor VIII:C (r=0.72, P less than 0.01). Four mild von Willebrand patients gave similar results by immunoassay and procoagulant assay methods. A previously identified patient with cross reacting material (CRM+) gave an immunoassay within the normal range (66%) with only 4% VIII:C activity detectable. The method offers a simple, sensitive and apparently reliable procedure for the assay of plasma factor VIII:C Ag which may prove useful in the further investigation of factor VIII:C Ag and antibody heterogeneity. The procedure offers an alternative to immunoradiometric assay and may be of potential use in the assessment of the haemophilia carrier state and possibly the early detection of thrombosis.

This study establishes a convenient method for screening plasma samples for abnormalities of the carbohydrate content of the factor VIII (FVIII) molecule. A radioimmuno-electrophoretic technique has been developed to quantitate the percentage binding of FVIII-related antigen (VIII-Ag) to the lectin concanavalin A (Con A). Plasma samples were electrophoresed through a strip of agarose containing Con A into agarose containing a mixture of unlabelled anti-FVIII and 125I-anti-FVIII where precipitant lines formed, the height of which was dependent upon the degree of VIII-Ag binding to Con A in the first gel. Using this system reduced binding of VIII-Ag to Con A was found in the plasma of 12 patients with moderate classical von Willebrand's disease (vWd), while the Con A binding of six haemophilia A patients fell within the normal range. The VIII-Ag in normal cryoprecipitate showed increased % binding to Con A while the VIII-Ag remaining in the cryo-supernate demonstrated reduced Con A % binding.

We describe an enzyme-immunoassay for the determination of factor VIII antigen. After representation of the isolation of proteins the enzyme-immunoassay is presented. The principle of the method is the following: Test plasma is mixed with rabbit antibody in excess and incubated at 37 degrees C. The incubation mixture is added to polystyrene tubes, which are coated with human factor VIII. The rabbit antibody is available to adhere to factor VIII coating the tube and can be detected with an enzyme-labeled antibody to rabbit IgG. This method is sensitive to 7.8 . 10(-3) U/ml factor VIII antigen; the variation coefficient is 10.9%.

A distinct sub-group of von Willebrand's disease is characterized by an electrophoretically faster mobility of the factor VIII related antigen. Some of the physico-chemical properties of this variant antigen were investigated in this communication. The effect of temperature was tested by heating aliquots (0.5 ml) for 20 minutes at 45 degrees C, 56 degrees C and 65 degrees C. The variant was found to be denatured at 56 degrees C while the control was denatured at 65 degrees C. The effect of pH was tested by assessing the quantity (Laurell technique) and electrophoretic mobility (two dimensional immunoelectrophoresis) of the antigen in a variety of buffers ranging in pH from 7.0 to 9.5. The quantity of antigen was variable both among variants and controls and the electrophoretic mobility of the variant antigen was faster at all pH's. Molecular weight differences between the variant and controls were not detected since the chromatographic profile of the variant was similar to that of the controls in Sepharose 6 B using a 0.02 M Tris-NaCL buffer at pH 7.0. The affinity of the antigen for human antibody was heterogeneous although the variant exhibited less affinity for one of the human antibodies but not the other. The inhibitory effect was more pronounced in serum than in plasma. Purified IGG, however, did not show any inhibition, as the residual antigen assayed by the Laurell technique, was similar to the expected values. This would imply that non-IgG plasmatic factors could also play a part in the observed inhibition.

Four unrelated patients with a severe form of von Willebrand disease showed no detectable factor VIII-related antigen (VIIIR:AG) in either their plasma or their platelets. They received cryoprecipitate infusions, three patients in a single injection each and one every day for 9 days before and after surgery. Platelet VIIIR:Ag was studied at different times during and after transfusion using electroimmunoassay of platelet extracts and electron microscopy of the platelets incubated with anti-VIIIR:Ag antibodies coupled to peroxidase. No VIIIR-Ag was detected in or around the patients' platelets, although this antigen was detected in the circulating blood. These results that there was no VIIIR:Ag uptake from the plasma by the platelets and that platelet VIIIR:Ag came from megakaryocytes.

An enzyme immunoassay (EIA) system has been developed to measure factor VIII-related antigen (VIIIAGN). This assay gives similar results to the commonly used Laurell electroimmunodiffusion (EID) assay for VIIIAGN as shown by comparison of both techniques with samples from healthy controls, patients with hemophilia A, and patients with von Willebrand's disease. The assay also has a greater precision than the EID technique as demonstrated by multiple assays of aliquots of a single sample. The use of this EIA test for VIIIAGN is simple and employs inexpensive reagents and equipment. The use of expensive antisera is minimized. EIA for VIIIAGN has the advantage of increased sensitivity compared to Laurell EIA.

Plasma samples from patients with various types of von Willebrand's disease were subdivided into six patterns according to the electrophoretic mobility and shape of VIIIR: Ag on crossed immunoelectrophoresis (CIE): pattern 1 no precipitation are, pattern 2 normal mobility with low arc, arc, pattern 3 intermediate mobility with low arc, pattern 4 faster anodal mobility with low arc, pattern 5 normal mobility with normal are height, pattern 6 faster anodal mobility with normal are height. Of 62 patients, 14 had pattern 1, 6 pattern 2, 16 pattern 3, 12 pattern 4, 9 pattern 5, and 5 pattern 6. Large pore polyacrylamide-agarose gel-crossed immunoelectrophoresis (PAAG-CIE) of crude factor VIII fraction from cryoprecipitate revealed no arcs in patients with pattern 1, three arcs of reduced height in the patients with patterns 2 and 3, four arcs very similar to normal control in patients with pattern 5, and two arcs with fast anodal migration in the patients with patterns 4 and 6. Crude factor VIII fractions from normal cryosupernatant showed one low fast anodally migrating arc corresponding to the fourth arc of normal cryoprecipitate. No peak was seen in patients with pattern 1, and one low fast anodal arc similar to normal control was present in the patients with patterns 2, 3, 4, 5 and 6.

Plasmas having no detectable factor VIII-related antigen but moderate factor VIII coagulant were obtained from two unrelated dogs homozygous for von Willebrand's disease and with the severe clinical expression of the disease. when these plasmas were gel-filtered in a buffer at physiologic ionic strength, the factor VIII coagulant eluted in the bed volume as a single well-defined peak. Addition of protease inhibitors, including diisopropylfluorophosphate, did not change the elution pattern. Each plasma was then combined individually with plasmas from six different mutants of canine haemophilia, all of which had normal factor VIII-related antigen but no detectable factor VIII coagulant. The factor VIII coagulant elution profile of these combined plasma resembled that of normal canine plasma. Slightly over half of the recovered factor VIII coagulant coeluted in the void volume with the factor VIII-related antigen; the rest eluted as a second, distinct peak of lower molecular weight. These results demonstrated that part of the factor VIII coagulant of the von Willebrand plasmas had bound to the factor VIII-related antigen of the haemophilic plasmas. This finding supports the theory that factor VIII exists as a macromolecular complex of nonidentical components in normal citrated plasma.

Commercially available antiserum to factor VIII was used in several tests to determine whether it might serve as a reference between research laboratories involved in investigation of the factor VIII complex and whether the antiserum might be useful in the screening of large populations of patients with hereditary disorders of factor VIII. In Ouchterlony plates, the antiserum gave a single line of identity with concentrated factor VIII, cryoprecipitate, and human plasma. The antiserum was capable of inhibiting the ristocetin response of normal platelets. Testing antigenic factor VIII by the Laurell technic with the commercial antiserum on plasmas from normal and stressed normal controls, patients with von Willebrand's disease, patients with hemophilia A, and obligate carriers of hemophilia A gave diagnostic and reproducible results.

Heterogeneous factor VIII related antigen isolated from endothelial cell postculture medium was characterized. On crossed immunoelectrophoresis, slow moving less anodal populations of factor VIII related antigen molecules were more prominent in endothelial cell postculture medium than in plasma. The protein synthesized by endothelial cells appeared as two discrete protein bands of different molecular weight in sodium dodecyl sulfate polyacrylamide gels. In contrast the factor VIII related antigen isolated from plasma moved as a single protein band in an identical gel system. The factor VIII related antigen from endothelial cell postculture medium was immunoisolated using monospecific rabbit antibody to normal human plasma factor VIII related antigen, electrophoresed on sodium dodecyl sulfate polyacrylamide gels, radiolabeled with 125I, trypsinized and subjected to peptide mapping using two-dimensional high voltage electrophoresis and thin-layer chromatography. The two forms of endothelial cell factor VIII antigen had virtually identical peptide maps. These studies suggest that the molecular heterogeneity of the factor VIII related antigen system reflects polymeric associations of identical subunits. Circulation in vivo may alter the ratio of polymer subsets.

Immunological and immunofluorescent studies carried out on plasma and platelets of three cases of congenital factor XIII deficiency are reported. Two of these patients were originally thought to have normal factor XIII subunit S and no subunit A. However, repeated assays carried out using different lots of antiserum showed that in reality the patients lacked both subunit S and subunit A. The false positive finding was due to the presence of a anti-factor VIII contaminant in the antiserum originally used. The third patient had a normal subunit S and no subunit A. No factor XIII antigen was found by the indirect immunofluorescent technique in normal, factor XIII deficiency and von Willebrand's disease platelets. On the contrary, by using the non-monospecific antiserum a fluorescent pattern similar to that observed by using an anti-factor VIII antiserum, had been noted. On the basis of the data presented in this paper a tentative classification of factor XIII deficiency in two groups is proposed: Type I is characterized by the lack of both factor XIII subunit S and A. Type II is characterized by a normal subunit S and no subunit A. The need for a re-evaluation of published case of factor XIII deficiency by means of monospecific antisera is indicated.

A plasma protein required for the support of ristocetin-induced platelet aggregation was isolated from antihemophilic factor concentrate and radiolabeled with 125I. A double-antibody radioimmunoassay was developed, with use of specific rabbit anti-VIII related antigen serum and goat anti-rabbit globulin. The assay is sensitive, reproducible, and technically simple to perform. Values obtained in normal subjects ranged from 0.65 to 1.53 units, similar to our normal range for VIII coagulant activity (0.67-1.43 units). However, normal or increased values of VIII-related antigen were observed in VIII coagulant-deficient hemophiliacs. Also, concentrations of VII-related antigen significantly exceeded coagulant concentrations in several patients with liver disease or disseminated intravascular coagulation, or both. Of a broad selection of congenital coagulation disorders examined, only patients with von Willebrand's disease had decreased VIII-related antigen concentrations, and these corresponded to the lowered concentration of ristocetin cofactor in the patients. In three transfused patients, VII-related antigen values correlated with the concentration of the cofactor. Our results suggest that the radioimmunoassay of VIII-related antigen is a simple and valuable adjunct in the study of patients with clotting abnormalities.

Classification of the subtypes of von Willebrand's disease (vWd) has been based on a quantitative deficiency or an abnormal multimeric composition of von Willebrand factor (vWf). Although the co-deficiency of a second protein, von Willebrand's antigen II (vW AgII), had been previously recognized, its concentration in a relatively large number of normal individuals or patients with well-defined vWd variants had not been studied. The plasma from patients with type I, IIA, IIB, IIC, and III (severe) vWd was evaluated, and the concentrations of vW AgII and vWf were determined. Although patients with type I and III vWd had reduced levels of both proteins, the plasma vW AgII concentration was normal in patients with type II vWd. Analysis of the results indicates that type I and type II variants can be discriminated with greater than 80% accuracy by comparison of results of these two antigenic assays. The normal levels of vW AgII in type II variants suggest a possible difference in the pathophysiology of type I and type II vWd.

A rapid method for measuring factor VIII antigen in plasma is presented, based upon optical measurement of the rate of agglutination of a stirred suspension of latex particles coated with anti-factor VIII immunoglobulin, when mixed with plasma containing factor VIII antigen. A log-log relationship exists between factor VIII concentration and the reciprocal of the time required for a fixed absorbance change. The assay is rapid, requiring less than 3 min for normal plasma, involves stable reagents, and can be performed with conventional instrumentation. Antigen values in normal plasma a well as plasma from patients with von Willebrand's disease (angiohemophilia) correlate well with values measured by electroimmunoassay or by radioimmunoassay.

An enzyme immunoassay system basing on a competitive method has been developed to measure factor VIII related antigen (F. VIII R:Ag). A sufficient discrimination at low F. VIII R:Ag concentrations was gained. This method appears to be sensitive to 7,8 X 10(-3) U/ml F. VIII R:Ag showing an intraassay coefficient of variation (CV) of 0,11. In comparison to the commonly used Laurell electroimmunodiffusion assay for factor VIII significant less antisera per sample for the enzyme immunoassay technique is necessary.

An immunoradiometric assay (I.R.M.A.) for antihaemophilic factor (factor VIII) has been developed which is based on a human inhibitor to procoagulant factor VIII (FVIIIC) which arose in a polytransfused haemophiliac. The assay detected and measured the antigen related to FVIIIC (FVIIICAg) in normal plasma and serum but failed to detect any antigen in samples from five patients with severe haemophilia. In a sixth patient with severe haemophilia a non-parallel dose-response curve was obtained. FVIIICAg was also present in plasma and serum from six samples of cord blood and in two samples of fetal serum obtained after termination of pregnancy. The assay may, therefore, be used to measure FVIIICAg in small serum samples and provides a potential practical method for prenatal diagnosis of haemophilia.

von Willebrand's antigen II (vW AgII), a plasma and platelet antigen immunologically and biochemically distinct from factor-VIII-related antigen (VIIIR:Ag), is decreased in von Willebrand's disease. vW AgII is released from platelets during aggregation and clotting. The release of platelet vW AgII was studied in washed platelets following aggregation by thrombin, collagen, and adenosine diphosphate (ADP). Total platelet vW AgII was 3.39 U/10(9) platelets. Thrombin and collagen yielded release of 85% and 86% of platelet vW AgII, respectively, at the highest concentrations. Release of platelet vW AgII was correlated with the release of 5-hydroxytryptamine (5HT). Release of vW AgII by collagen and thrombin was inhibited by metabolic inhibitors. In addition, collagen release of vW AgII was inhibited by aspirin. In clinical syndromes associated with intravascular platelet destruction, marked elevations of plasma vW AgII were noted. Thus, vW AgII is released by a metabolically active process from platelets during aggregation. In addition, vW AgII appears to be a marker of intravascular platelet destruction.

Factor-VIII-related antigen was measured, both by an enzyme immunoassay using a microplate method and by the Laurell technique, in normal people, patients with von Willebrand's disease, haemophiliacs, and obligatory haemophilia carriers. The enzyme immunoassay was simpler to perform and gave equally reliable and reproducible results. Many more assays could be carried out at any one time.

We have localized factor-VIII-related antigen, using immunofluorescence and electron microscopy, in adult human blood vessels. In addition to its presence in endothelial cells, the antigen was localized within subendothelium and the layers of elastic lamina closest to the lumen. Also, we provide the first morphological evidence that factor-VIII-related antigen is associated with collagen fibrils within the vessel wall. These studies suggest that this subendothelial factor-VIII-related antigen may play a role in the adhesion of platelets to subendothelial components following endothelial injury.