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Platelet adhesion to fibrillar collagens (types I, II, III, and V) and nonfibrillar collagens (types IV, VI, VII, and VIII) was investigated in the presence of physiologic concentrations of divalent cations under conditions of stasis and flow. Under static conditions, platelet adhesion was observed to collagen types I through VII but not to type VIII. Under flow conditions, platelet adhesion to collagen types I, II, III, and IV was almost independent of shear rates above 300/s. Collagen type V was nonadhesive. Platelet adhesion to collagen type VI was shear rate-dependent and optimal at a rate of 300/s. Collagen types VII and VIII showed minor reactivity and supported platelet adhesion only between shear rates 100 to 1,000/s. Monoclonal antibody (MoAb) 176D7, directed against platelet membrane glycoprotein Ia (GPIa; very late antigen [VLA]-alpha 2 subunit), completely inhibited platelet adhesion to all collagens tested, under conditions of both stasis and flow. Platelet adhesion to collagen type III at shear rate 1,600/s was only inhibited for 85%. The concentration of antibody required for complete inhibition of platelet adhesion was dependent on the shear rate and the reactivity of the collagen. An MoAb directed against GPIIa (VLA-beta subunit) partially inhibited platelet adhesion to collagen. These results show that GPIa-IIa is a major and universal platelet receptor for eight unique types of collagen.

The aim of this investigation was to identify domains of collagen type I that can support platelet adhesion under flow conditions. Four cyanogen bromide (CB) fragments composing 87% of the collagen alpha 1(I)-chain were studied under static and flow conditions. Under static conditions, bovine and human collagen fragment alpha 1(I)CB3 induced aggregate formation, whereas alpha 1(I)CB7 and alpha 1(I)CB8 supported adhesion of dendritic and contact platelets. Bovine alpha 1(I)CB6 weakly supported platelet adhesion. At shear rate 300/s, collagen fragment alpha 1(I)CB3 strongly supported platelet adhesion, whereas lower platelet adhesion was observed to alpha 1(I)CB7 and alpha 1(I)CB8. The fragment alpha 1(I)CB6 did not support platelet adhesion under flow conditions. Adhesion to alpha 1(I)CB3 was completely inhibited by a low concentration (0.6 IgG microgram/mL) of anti-GPIa monoclonal antibody (MoAb), whereas this concentration of antibody partially inhibited adhesion to alpha 1(I)CB7 and alpha 1(I)CB8. At higher concentrations (3 micrograms/mL) the anti-glycoprotein Ia (GPIa) antibody completely inhibited adhesion to alpha 1(I)CB8 and further reduced adhesion to alpha 1(I)CB7. Platelet adhesion to alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 was strongly inhibited by an anti-GPIb MoAb. A MoAb against the GPIb-binding site of von Willebrand factor (vWF) strongly inhibited platelet adhesion to alpha 1(I)CB7 and alpha 1(I)CB8, whereas platelet adhesion to alpha 1(I)CB3 was not inhibited. We conclude that under flow conditions alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 support GPIa/IIa-dependent platelet adhesion. The GPIb-vWF interaction is important under flow conditions for adhesion to alpha 1(I)CB7 and alpha 1(I)CB8 and probably also to alpha 1(I)CB3.

The aim of this investigation was to identify domains of collagen type I that can support platelet adhesion under flow conditions. Four cyanogen bromide (CB) fragments composing 87% of the collagen alpha 1(I)-chain were studied under static and flow conditions. Under static conditions, bovine and human collagen fragment alpha 1(I)CB3 induced aggregate formation, whereas alpha 1(I)CB7 and alpha 1(I)CB8 supported adhesion of dendritic and contact platelets. Bovine alpha 1(I)CB6 weakly supported platelet adhesion. At shear rate 300/s, collagen fragment alpha 1(I)CB3 strongly supported platelet adhesion, whereas lower platelet adhesion was observed to alpha 1(I)CB7 and alpha 1(I)CB8. The fragment alpha 1(I)CB6 did not support platelet adhesion under flow conditions. Adhesion to alpha 1(I)CB3 was completely inhibited by a low concentration (0.6 IgG microgram/mL) of anti-GPIa monoclonal antibody (MoAb), whereas this concentration of antibody partially inhibited adhesion to alpha 1(I)CB7 and alpha 1(I)CB8. At higher concentrations (3 micrograms/mL) the anti-glycoprotein Ia (GPIa) antibody completely inhibited adhesion to alpha 1(I)CB8 and further reduced adhesion to alpha 1(I)CB7. Platelet adhesion to alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 was strongly inhibited by an anti-GPIb MoAb. A MoAb against the GPIb-binding site of von Willebrand factor (vWF) strongly inhibited platelet adhesion to alpha 1(I)CB7 and alpha 1(I)CB8, whereas platelet adhesion to alpha 1(I)CB3 was not inhibited. We conclude that under flow conditions alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 support GPIa/IIa-dependent platelet adhesion. The GPIb-vWF interaction is important under flow conditions for adhesion to alpha 1(I)CB7 and alpha 1(I)CB8 and probably also to alpha 1(I)CB3.

Endothelial cells (EC) synthesize and secrete von Willebrand factor (vWF), a multimeric glycoprotein required for normal hemostasis. Within human endothelial cells, vWF multimers of extremely high molecular weight are stored in rod-shaped organelles known as Weibel-Palade bodies. Inflammatory mediators, such as interleukin-1, induce in vitro a variety of procoagulant responses by EC, including the secretion of stored vWF. We postulated that other inflammatory mediators might act to balance this procoagulant reaction, thereby assisting in the maintenance of blood fluidity during immune activation. Both gamma- interferon (gamma-IFN) and tumor necrosis factor (TNF) were found to act independently and cooperatively to depress the stimulated release of vWF from EC. Analysis of stored vWF in either gamma-IFN and/or TNF- treated EC demonstrated a loss of high molecular weight multimers while immunofluorescent studies documented a loss of visible Weibel-Palade bodies. This suggests that gamma-IFN and TNF interfere with normal vWF storage. gamma-IFN acted in a dose-, time-, and RNA-dependent fashion, and its inhibition of vWF release was reversible with time. No effect of gamma-IFN on EC was noted when anti-serum to gamma-IFN was added. Unlike gamma-IFN, alpha-interferon did not effect EC vWF. Therefore, gamma-IFN and TNF may be important in decreasing vWF release during inflammatory or immunologic episodes.

The factor VIII/von Willebrand factor (f.VIII/vWf) protein was purified from the plasma of a patient with von Willebrand's disease (vWd). The patient had all of the classic laboratory findings of vWd except for the ristocetin-induced platelet aggregation of his own platelet-rich plasma. The disease has been documented in three generations. Comparison of the purified normal and vWd f.VIIi/vWf protein revealed several abnormalities, including decreased concentration of f.VIII/vWf antigen; decreased specific vWf activity; absence of the larger molecular forms of the f.VIII/vWf protein; carbohydrate deficiencies affecting the sialic acid, penultimate galactose and N- acetylglucosamine moieties; and decreased binding of the f.VIII/vWf protein to its platelet receptor. These studies indicate the multiplicity of biochemical and functional abnormalities associated with the f.VIII/vWf protein in vWd. f.VIII/vWf protein to normal f.VIII/vWf protein that had been treated with 2-mercaptoethanol (2-ME) to reduce the multimer size and then treated with specific exoglycosidases to remove the sialic acid and penultimate galactose residues revealed similar biologic properties.

A bleeding diathesis is described which is phenotypically indistinguishable from hemophilia A and which has been transmitted as a dominant trait in three generations of women in a North Carolina kindred. The abnormal phenotype is characterized by clinical mildness and slightly abnormal clotting time, prothrombin consumption, and partial thromboplastin time. Bleeding time, platelet count, clot retraction, tourniquet test, and prothrombin time are normal. Concentration of factors I, II, V, VII, IX, X, and XII are normal, while factor VIII activity is reduced to 2%-5% of control values. De novo synthesis of factor VIII does not occur after transfusion; factor VIII-related antigen is normal; patients'plasmas aggregate platelets normally in the presence of ristocetin, and a typical protein pattern is seen when a chymotryptic digest of cryoprecipitate of the proband is examined by SDS-polyacrylamide gel electrophoresis. Six possible genetic explanations are entertained. Balanced X-autosomal translocation of hemophilia A heterozygotes has been excluded by cytogenetic analysis of metaphase chromosomes. Classes von Willebrand's disease (vWd) is probably excluded on the basis of the laboratory data, and extreme lyonization of hemophilia A heterozygotes on probabilistic grounds. The genetic possibilities which cannot be excluded include a previously unrecognized variant mutation at the vWd locus, a dominant mutation at the hemophilia A locus on the X chromosome, and dominant mutation at a hypothetical fourth locus involved in factor VIII synthesis and control.

The adhesive glycoproteins laminin, thrombospondin, and von Willebrand factor bind specifically and with high affinity to sulfated glycolipids. These three glycoproteins differ, however, in their sensitivity to inhibition of binding by sulfated monosaccharides and polysaccharides. Heparin strongly inhibits binding of thrombospondin but only weakly inhibits binding of laminin and von Willebrand factor. Fucoidan strongly inhibits binding of both laminin and thrombospondin but not of von Willebrand factor. Laminin shows significant specificity for inhibition by monosaccharides, whereas thrombospondin does not. Thus, specific spacial orientations of sulfate esters may be primary determinants of binding for the three proteins. Laminin, thrombospondin, and von Willebrand factor also differ in their relative binding affinities for purified sulfated glycosphingolipids. The three proteins strongly prefer terminal-sulfated lipids and bind only weakly to sulfated gangliotriaosyl ceramide with a sulfate ester on the penultimate galactose. Thrombospondin binds with highest affinity to galactosyl sulfatide but only weakly to more complex sulfatides, whereas von Willebrand factor prefers galactosyl sulfatide but binds with moderate affinity to various sulfated glycolipids. Laminin also is less selective than thrombospondin but is less sensitive for detection of low sulfatide concentrations. Galactosyl sulfatide at 1-5 pmol can be detected by staining of lipids separated on high performance TLC with 125I-thrombospondin or 125I-von Willebrand factor. 125I-von Willebrand factor was examined as a reagent for detecting sulfated glycolipids in tissue extracts. Rat kidney lipids contain 5 characterized sulfated glycolipids: galactosyl ceramide I3-sulfate, lactosyl ceramide II3-sulfate, gangliotriaosyl ceramide II3-sulfate, and bis-sulfated gangliotriaosyl and gangliotetraosyl ceramides. von Willebrand factor detects all of these lipids as well as several additional minor sulfated lipids. Complex monosulfated lipids are detected in several human tissues including kidney, erythrocytes, and platelets by this technique.

Five patients with glycogen storage disease type I (GSD-I) were evaluated for a bleeding diathesis and subsequently were given an infusion of 1-deamino-8-D-arginine vasopressin (DDAVP). Although platelet counts were normal or slightly elevated, the baseline template bleeding times were prolonged in four of the patients. Prothrombin times and activated partial thromboplastin times were normal, while ADP- and epinephrine-induced platelet aggregations were absent in the three patients tested. Ristocetin- and collagen-induced platelet aggregations were abnormal. Laurell and immunoradiometric determinations of the factor VIII-related antigen (vWf antigen) were decreased. Glyoxyl agarose gel electrophoresis of the patients' plasma revealed abnormal multimer patterns in four of the five patients. After the DDAVP infusion the platelet aggregation abnormalities persisted; however, the bleeding time and the von Willebrand antigen and activity corrected. We conclude that GSD-Ia patients may have a metabolically acquired form of von Willebrand's syndrome as well as an acquired intrinsic platelet defect, and that DDAVP may be useful in the management of bleeding in these patients.

Synthetic peptides containing the adhesion site recognition sequences present on the A alpha and gamma chains of fibrinogen were studied for their effect on the binding of endogenous platelet-von Willebrand factor (vWF) and exogenous plasma-vWf to thrombin-stimulated platelets. In agreement with previously reported data, the tetrapeptide consisting of the RGDS sequence was a more potent inhibitor of plasma-vWf binding to platelets than was the pentadecapeptide of the carboxy terminus of the fibrinogen gamma-chain (IC50 10.6 mumol/L for the RGDS tetrapeptide v 44.9 mumol/L for the gamma-chain pentadecapeptide). No apparent synergy in the inhibition of plasma-vWf binding was noted when the RGDS and gamma-chain peptides were used together (IC50 15.2 mumol/L). In contrast, the gamma-chain peptide was significantly more inhibitory than was the RGDS tetrapeptide on the binding of platelet-vWf to platelets (IC50 1.4 mumol/L for the gamma-chain pentadecapeptide v 4.5 mumol/L for the RGDS tetrapeptide, P less than .05), and there was significant synergy in the inhibition of platelet-vWf binding noted when the gamma-chain and RGDS peptides were used together (IC50 0.04 mumol/L). These results indicate that the binding of platelet-vWf to its receptor on the platelet glycoprotein IIb/IIIa complex involves both the RGDS and gamma-chain recognition sites. In contrast to the results with plasma-vWf binding, the gamma-chain recognition site appears to be more important than the RGDS recognition site in platelet- vWf binding to platelets.

Two monoclonal antibodies--one that blocks ristocetin-induced platelet binding of von Willebrand factor to glycoprotein Ib and one that blocks adenosine diphosphate-induced binding of fibrinogen to the glycoprotein IIb/IIIa complex--were used to assess the binding site(s) for von Willebrand factor when platelets are stimulated with thrombin or adenosine diphosphate (ADP). Neither agonist induced binding of von Willebrand factor to glycoprotein Ib. ADP and thrombin induced von Willebrand factor binding exclusively to the glycoprotein IIb/IIIa complex. The results of the site of binding of von Willebrand factor with thrombasthenic platelets were consistent with the data obtained with the monoclonal antibodies and normal platelets. Human fibrinogen caused complete inhibition of thrombin-induced von Willebrand factor binding to normal platelets at concentrations considerably below that found in normal plasma. We conclude that thrombin induces very little binding of exogenous von Willebrand factor to platelets at normal plasma fibrinogen levels.

Studies of the prothrombin time (PT) have revealed that contact with borosilicate or commercial siliconized borosilicate markedly shortens the PT. This shortening is related to the activation of the contact phase of blood coagulation. This shortening of the PT occurs in both normal whole blood and plasma when stored in borosilicate or siliconized borosilicate tubes at 4 degree C and to a lesser degree at room temperature. Studies indicated the importance of several coagulation factors in decreasing the PT. The PT did not change in blood deficient in factor XII or in plasma deficient in Fletcher factor or high molecular weight kininogen, while blood deficient in CI esterase inhibitor (CI INH) had the most profound shortening. Shortening of the PT correlated directly with increased levels of factor VII. When purified CI INH was added to normal blood, it markedly reduced the activation of factor VII and the shortening of the PT in a dose-related manner. These studies indicate the pivotal roles of the contact phase of coagulation in initiating activation of the PT and of CI INH in inhibiting the activation of the coagulation factor(s) responsible for the cold-promoted activation of factor VII.

Quantitative ristocetin-induced platelet aggregation of normal platelet- rich plasma (PRP) decreased with time after PRP preparation. An increase in p H of the PRP with time proved to be responsible for this finding. Diffusion of CO2from the plasma is the prime determinant of the change in pH. Since a complex combination of factors influences CO2 diffusion (surface area-to-volume relationship, capping, mixing, etc.) The change in pH is variable with time. Thus, quantitative ristocetin aggregation should be pH controlled. A simple device for maintaining PRP pH constant by control of the ambient pCO2 was designed and found effective in keeping both pH and quantitative ristocetin aggregation constant over a prolonged period of time. It can be adapted for use in platelet aggregation studies employing other reagents. The pH dependence of ristocetin-induced platelet aggregation is consistent with other data supporting an elctrostatic interaction between the platelet, von Willebrand factor, and ristocetin. We favor a model wherein ristocetin neutralizes some of the platelet's negative change and permits the von Willebrand factor to bridge sites on separate platelets to induce agglutination.

The factor VIII/von Willebrand factor protein was radiolabeled after modification by galactose oxidase and reduction with tritiated potassium borohydride. This mild efficient method for labeling resulted in retention of over 90% of the biologic activities of the factor VIII/von Willebrand factor protein. Binding of this protein to platelets was found to be specific, and binding sites could be saturated in the presence of ristocetin. However, binding was highly dependent on ristocetin concentration, as the number of human factor VIII/von Willebrand factor molecules bound per platelet was a function of the ristocetin concentration. At a ristocetin concentration of 0.55 mg/ml, each platelet binds approximately 11,000 factor VIII/von Willebrand factor molecules per platelet. Scatchard analysis of the concentration-dependent binding sites yielded a hyperbolic plot that appeared to be related to the existence of two classes of binding sites. The higher affinity class had a Kd of 3.7 x 10(-10) M 3500 sites/platelet, while the lower affinity class had a Kd of 2.35 x 10(- 9) M and a capacity of 7500 sites/platelet. As with ristocetin-induced platelet agglutination, the carbohydrate content plays a significant role in the binding of the factor VIII/von Willebrand factor protein to the platelet.

Plasma exchange has been proposed as a treatment for multiple disorders. Three patients with amyotropic lateral sclerosis, who were hemostatically normal, were studied through a total of 11 4-liter exchanges. Plasma was replaced by an equal volume of 5% albumin or 5% plasma protein fraction. Serial studies revealed that immediately after the exchange transfusion, there was significant prolongation of the prothrombin, partial thromboplastin, and thrombin times with reduction of the fibrinogen and antithrombin III levels. Factors V, VII-X, IX, and X were all significantly decreased, as were the factor VIII antigen, procoagulant, and the ristocetin cofactor activities. Platelet counts were obtained before and after exchanges and revealed significant decreases. Four hours after exchange, all parameters remained abnormal except the factor IX, ristocetin cofactor, and factor VIII procoagulant activities. By 24 hr, all hemostatic parameters had returned to normal. These studies indicate that plasma-exchange transfusion with material devoid of coagulation factors results in a coagulation defect that may be of clinical significance in a hemostatically compromised patient.

A bleeding diathesis is described which is phenotypically indistinguishable from hemophilia A and which has been transmitted as a dominant trait in three generations of women in a North Carolina kindred. The abnormal phenotype is characterized by clinical mildness and slightly abnormal clotting time, prothrombin consumption, and partial thromboplastin time. Bleeding time, platelet count, clot retraction, tourniquet test, and prothrombin time are normal. Concentration of factors I, II, V, VII, IX, X, and XII are normal, while factor VIII activity is reduced to 2%-5% of control values. De novo synthesis of factor VIII does not occur after transfusion; factor VIII-related antigen is normal; patients'plasmas aggregate platelets normally in the presence of ristocetin, and a typical protein pattern is seen when a chymotryptic digest of cryoprecipitate of the proband is examined by SDS-polyacrylamide gel electrophoresis. Six possible genetic explanations are entertained. Balanced X-autosomal translocation of hemophilia A heterozygotes has been excluded by cytogenetic analysis of metaphase chromosomes. Classes von Willebrand's disease (vWd) is probably excluded on the basis of the laboratory data, and extreme lyonization of hemophilia A heterozygotes on probabilistic grounds. The genetic possibilities which cannot be excluded include a previously unrecognized variant mutation at the vWd locus, a dominant mutation at the hemophilia A locus on the X chromosome, and dominant mutation at a hypothetical fourth locus involved in factor VIII synthesis and control.

The characteristics of the intact factor VIII/von Willebrand factor protein binding to human platelets was compared to 2-mercaptoethanol- treated factor VIII/von Willebrand factor protein and to fractions of plasma factor VIII/von Willebrand factor protein that elute after the void volume. These studies indicate that the factor VIII/von Willebrand factor protein larger size oligomers bind preferentially with high affinity to low capacity sites on human platelets. The intermediate and smaller size oligomers bind with intermediate or low affinity to sites with a much greater capacity. The results from binding analysis are also paralleled by the competitive inhibition of the intact factor VIII/von Willebrand factor protein by the various 2-mercaptoethanol- treated materials. These studies indicate that the two classes of binding sites seen in previous reports of factor VII/von Willebrand factor binding reflect heterogeneity in the oligomer size of the factor VIII/von Willebrand factor protein used in these assays. This study provides a model for understanding some of the normal structure- function relationships of the normal factor VIII/von Willebrand factor protein and the defect(s) in a variant form of von Willebrand's disease. In this form of the disease, decreased factor VIII/von Willebrand factor binding to platelets is reflected in decreased von Willebrand factor activity but coagulant and/or antigen levels are normal or only slightly decreased.

We have studied the platelet glycoproteins (GP) GPIb and the GPIIb/IIIa and the expression of alpha granule proteins (AGP) on the platelet (P) surface following thrombin (T) stimulation. The platelets were separated from plasma proteins on a arabinogalactan gradient. The P were stimulated with purified alpha T 0.1u/108p. Either monospecific polyclonal or murine monoclonal antibodies were used to detect the P glycoprotein and AGP. The platelets were analyzed on an EPICS V Flow Cytometer. Resting P had small amounts of AGP (2-8%) present on their surface. Within 1-3 min. after T stimulation significantly increased amounts of PF4 (26%) vWf (8%) Ig (10%) and the 140 kD alpha granule membrane (70%) were present on the P surface. The peak expression of all the AGP occurred within 5 mins. The 140 kD activation protein remained stable over 3-60 mins, in contrast the PF4 and the vWf expression peaked at 5 mins. and then decreased to near baseline levels. The GPIb and GPIIb/IIIa showed different patterns after activation. The GPIb intensity and number of positive cells decreased over time, while the GPIIb/ IIIa increased in flourescent intensity and the number of positive cells. These studies indicate that T stimulation of AGP on the P surface. vWf and P4 have a transient appearance on the P surface while Ig and the 140 kD activation protein both appear to become stable components of the P plasma membrane. This technique of detecting platelet activation is a specific, sensitive, and rapid method.