Abnormal VIII: von Willebrand factor patterns in the plasma of patients with the hemolytic-uremic syndrome

Plasma VIII:von Willebrand factor antigen (VIII:vWF) levels were elevated approximately two- to eightfold in seven patients (three adults and four children) during acute episodes of thrombocytopenia, renal failure, and hemolytic anemia (the hemolytic-uremic syndrome, HUS). In all seven patients, there was an alteration in plasma VIII:vWF patterns during these acute HUS episodes, so that the largest VIII:vWF forms were relatively decreased. Plasma VIII:vWF multimer patterns returned to normal, or nearly to normal, as platelet counts returned to preexisting levels, even in the patients whose recovery of renal function was incomplete and whose plasma VIII:vWF antigen level remained above normal. The sister of one of the HUS patients had a similar clinical prodrome (gastroenteritis) that was not followed by thrombocytopenia or renal failure and was not accompanied by an elevated level or abnormal forms of plasma VIII:vWF. These results suggest that an alteration in VIII:vWF metabolism, distribution, or interaction with platelets is associated with acute HUS episodes. In contrast to patients with chronic relapsing thrombotic thrombocytopenic purpura, none of the HUS patients (either during or after the acute HUS episodes) had a defect in the conversion of unusually large VIII:vWF multimers derived from endothelial cells to the VIII:vWF forms found in normal plasma.     

A new von Willebrand variant (type I, New York): increased ristocetin-induced platelet aggregation and plasma von Willebrand factor containing the full range of multimers

We report three members of a family who had reduced levels of plasma von Willebrand factor (vWF) and increased ristocetin-induced platelet aggregation (RIPA) (aggregation of platelet-rich plasma with ristocetin at a concentration of 0.45 mg/mL), as previously reported in type IIB and pseudo-von Willebrand's disease (vWD). However, in contrast to the latter two disorders in which the larger vWF multimers are absent in plasma, the entire range of vWF multimers was observed in the patients' plasma after sodium dodecyl sulfate-agarose gel electrophoresis, and all vWF multimers (including the largest) were present in the same proportion as in normal plasma and type I vWD. Thus, despite increased RIPA, the levels and multimeric pattern of vWF in this family's plasma were indistinguishable from those in type I vWD in which RIPA is usually decreased. Addition of ristocetin to the patients' platelet- rich plasma resulted in the removal of vWF (and, more selectively, of the large multimers) at lower concentrations of ristocetin than normal, as in type IIB and pseudo-vWD. The defect in the patients was localized to their vWF, which had an enhanced capacity for aggregating washed normal platelets in the presence of low concentrations of ristocetin and for aggregating pseudo-vWD platelets (in the absence of ristocetin). Both glycoproteins (GP) Ib and IIb-IIIa were involved in the enhanced aggregation response. RIPA (at low ristocetin concentrations) in the patients' platelet-rich plasma was abolished by a monoclonal antibody (AP1) to GPIb and was markedly reduced by monoclonal antibodies (10E5 and LJP9) that block adenosine diphosphate and thrombin-induced binding of vWF and fibrinogen to GPIIb-IIIa but was unaffected by an antibody (LJP5) that only blocks vWF binding. Partial inhibition of the initial aggregation slope (and complete inhibition of second phase aggregation) was achieved with creatine phosphate/creatine phosphokinase. EDTA blocked second-phase aggregation but was without effect on the initial slope. The findings in this family combine some features of both type I vWD (normal pattern of vWF multimers in plasma) and type IIB vWD (increased RIPA) and further demonstrate the increasing complexity of the structure-function relationships in vWD.     

Aurin tricarboxylic acid: a novel inhibitor of the association of von Willebrand factor and platelets

Shear stress activated platelets undergo aggregation in the presence of large or unusually large von Willebrand factor (vWF) multimers without the addition of ristocetin or any other exogenous chemical. This phenomenon may be analogous to the platelet aggregation that leads to thrombosis in the narrowed arteries and arterioles of patients with atherosclerosis or vasospasm. A triphenyl-methyl compound, aurin tricarboxylic acid (ATA), inhibits shear-induced, vWF-mediated platelet aggregation in platelet-rich plasma (PRP) in concentrations above 200 mumol/L and in buffer suspensions of washed platelets at a concentration of 0.1 mumol/L. In a concentration-dependent manner, ATA also inhibits ristocetin-induced, vWF-mediated platelet clumping in both fresh and formaldehyde-fixed platelet suspensions. This inhibition can be overcome by increasing the concentration of vWF, following the kinetics of first order competitive inhibition. ATA prevents the attachment to platelets of the largest vWF multimeric forms found in normal plasma and of the unusually large vWF multimers derived from endothelial cells. The rate of aggregation and degree of inhibition by ATA is not accounted for by the binding of ristocetin or calcium. Arachidonic acid- and adenosine diphosphate (ADP)-induced aggregation are not inhibited by ATA. Platelets incubated with ATA can be easily separated from the compound. However, ATA binds to large vWF multimeric forms and inhibits their ristocetin-induced interaction with platelet glycoprotein Ib. Because ATA also inhibits shear-induced, vWF-mediated platelet aggregation in vitro in the absence of ristocetin, it may be a useful prototype compound to impede the development of arterial thrombosis in vivo.     

Interaction of purified type IIB von Willebrand factor with the platelet membrane glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and initiates aggregation.

Von Willebrand factor (vWF) was purified from the plasma of a patient with type IIB von Willebrand disease (vWF from such a patient, IIB vWF) who had a normal platelet count and showed no evidence of spontaneous platelet aggregation. Large multimers of IIB vWF were absent from purified preparations and from plasma. Ristocetin-induced platelet aggregation was enhanced by purified IIB vWF. The aggregation of washed normal platelets mixed with IIB vWF (0.4 microgram/ml) required lower amounts of ristocetin than the aggregation of normal platelets mixed with the same concentrations of normal vWF. Moreover, purified IIB vWF alone induced aggregation of platelet-rich plasma at concentrations as low as 10 micrograms of IIB vWF/ml in the absence of any other agonist. Aggregation was blocked by a monoclonal antibody against the platelet membrane glycoprotein, GPIb, as well as by an anti-GPIIb/IIIa antibody. Washed platelet suspensions were promptly aggregated by IIB vWF only when fibrinogen and CaCl2 were added to the mixture. Purified IIB vWF induces the binding of fibrinogen to platelets. Such binding was blocked by the anti-GPIb monoclonal antibody as well as by the anti-GPIIb/IIIa monoclonal antibody that inhibited aggregation. A second anti-GPIIb/IIIa antibody, which has the property of blocking vWF but not fibrinogen binding to platelets, blocked neither aggregation nor fibrinogen binding induced by IIB vWF. These studies demonstrate that platelet aggregation is triggered by the initial interaction of IIB vWF with GPIb which is followed by exposure of fibrinogen binding sites on GPIIb/IIIa. Fibrinogen binds to these sites and acts as a necessary cofactor for the aggregation response.     

Enhanced ristocetin-induced von Willebrand factor binding to platelet glycoprotein Ib in patients with steroid-responsive nephrotic syndrome

To elucidate the mechanism of enhanced ristocetin-induced platelet aggregation (RIPA) in steroid-responsive nephrotic syndrome (SRNS), plasma levels of von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor (RCof) were examined in 6 patients and the amount of ristocetin-induced vWF binding to platelets was determined. At the initial or relapse stage, the plasma vWF:Ag level was 415 +/- 137% and the RCof level was 364 +/- 117%. The ratio of RCof/vWF:Ag was 0.90 +/- 0.15 and no abnormalities of vWF:Ag multimers were observed, indicating that neither functional nor structural abnormalities were present in patient's plasma. The amount of ristocetin-induced normal vWF binding to nephrotic washed platelets, when ristocetin was used at concentrations of 0.5, 0.75, and 1.0 mg/ml, was 152-163% above the binding to normal platelets. In addition, nephrotic washed platelets resuspended in either normal or nephrotic plasma aggregated at a low concentration of ristocetin (0.75 mg/ml) which did not induce aggregation of normal platelets. In accordance with these observations, the decrease of Alcian blue 8GX binding to platelets, reflecting diminished surface negative charge, was also observed. These results appear to indicate that the plasma vWF level and the altered surface-negative charge in platelets both contribute to heightened vWF binding to GPIb, thus lowering the ristocetin concentration required for RIPA in SRNS.     

Platelet--von Willebrand factor interactions in type IIB von Willebrand's disease

Type IIB von Willebrand's disease (vWD) is a distinct form of this disorder in which the largest multimers of the von Willebrand factor (vWF) are lacking in plasma but present in platelets. When the vasopressin analogue, 1-deamino-8-D-arginine vasopressin (DDAVP), is given to patients with type IIB vWD, an abnormal vWF is released to plasma. This vWF causes thrombocytopenia in vivo and platelet aggregation in vitro. Aggregation occurs in the plasma milieu and thus at physiological fibrinogen concentration. In this study we demonstrate that IIB post-DDAVP vWF aggregated only metabolically active platelets. The platelet aggregation was completely inhibited by EDTA and PGE1, and either inhibited or greatly weakened by ASA, demonstrating the role of divalent cations and thromboxane A2 formation. In spite of inhibiting platelet aggregation, EDTA, PGE1 and ASA did not prevent platelet binding of IIB post-DDAVP vWF. An antiserum against GP Ib made normal platelets less responsive to the IIB vWF although neither platelet aggregation nor vWF binding were completely prevented. The aggregation was fibrinogen-dependent and platelets from patients with Glanzmann's thrombasthenia were unresponsive. The studies provide evidence that IIB post-DDAVP vWF is bound to unstimulated platelets and that the interaction between vWF and platelets in type IIB vWD is different from ristocetin-induced as well as thrombin- and epinephrine-induced binding to platelets of normal vWF.     

Enhanced proteolysis of plasma von Willebrand factor in thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome

To examine whether enhanced in vivo proteolysis of von Willebrand factor (vWF) would account for the reported loss of larger multimers in acute thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS), we studied eight patients with acute TTP/HUS whose blood samples were collected into an anticoagulant containing a cocktail of protease inhibitors to impede in vitro proteolysis. In all, enhanced proteolytic degradation of vWF was expressed as a relative decrease in the intact 225-Kd subunit of vWF and a relative increase in the 176-Kd fragment. However, instead of the loss of larger forms of normal multimers reported by other investigators, the plasma of all but one of our patients (whether they had TTP or HUS) contained a set of larger than normal (supranormal) multimers. Hence, although proteolytic fragmentation of vWF was enhanced during acute TTP/HUS, this phenomenon was not associated with the loss of larger multimers. In the five patients who survived the acute disease and underwent plasma exchange (three with HUS and two with chronic relapsing TTP), subunits and fragments returned to normal values, and supranormal multimers were no longer detected in plasma. In conclusion, even though vWF proteolysis is enhanced in acute TTP/HUS, it does not lead to loss of larger multimers.     

Shear-induced platelet aggregation can be mediated by vWF released from platelets, as well as by exogenous large or unusually large vWF multimers, requires adenosine diphosphate, and is resistant to aspirin

Fluid shear stress in arteries and arterioles partially obstructed by atherosclerosis or spasm may exceed the normal time-average level of 20 dyne/cm2. In vitro, at fluid shear stresses of 30 to 60 dyne/cm2 applied for 30 seconds, platelet aggregation occurs. At these shear stresses, either large or unusually large von Willebrand factor (vWF) multimers in the suspending fluid exogenous to the platelets mediates aggregation. Adenosine diphosphate (ADP) is also required and, in these experiments, was released from the platelets subjected to shear stress. At 120 dyne/cm2, the release of endogenous platelet vWF multimers can substitute for exogenous large or unusually large vWF forms in mediating aggregation. Endogenous released platelet vWF forms, as well as exogenous large or unusually large vWF multimers, must bind to both glycoproteins Ib and the IIb/IIIa complex to produce aggregation. Shear- induced aggregation is the result of shear stress alteration of platelet surfaces, rather than of shear effects on vWF multimers. It is mediated by either large plasma-type vWF multimers, endogenous released platelet vWF forms, or unusually large vWF multimers derived from endothelial cells, requires ADP, and is not inhibited significantly by aspirin. This type of aggregation may be important in platelet thrombus formation within narrowed arterial vessels, and may explain the limited therapeutic utility of aspirin in arterial thrombosis.     

Shear-induced platelet aggregation requires von Willebrand factor and platelet membrane glycoproteins Ib and IIb-IIIa

Different types of platelets in various types of plasma were subjected to levels of shear stress that produce irreversible platelet aggregation in normal platelet-rich plasma (PRP). At shear stresses of 90 or 180 dyne/cm2 applied for 30 seconds or five minutes, aggregation was either absent or only transient and reversible using severe von Willebrand's disease (vWD) PRP (less than 1% von Willebrand factor, vWF); Bernard-Soulier syndrome (BSS) PRP (platelets deficient in the membrane glycoprotein Ib, GPIb); normal PRP plus monoclonal antibody (MoAb) to GPIb; thrombasthenic PRP (platelets deficient in membrane glycoprotein IIb-IIIa complex, GPIIb-IIIa); and normal PRP plus MoAb to GPIIb-IIIa. Shear-induced aggregation was inhibited under the above conditions, even though the platelets were activated to release their granular contents. Sheared normal platelets in vWD plasma aggregated in response to added vWF. These studies demonstrate that the formation of stable platelet aggregates under conditions of high shear requires vWF and the availability of both GPIb and GPIIb-IIIa on platelet membranes. The experiments demonstrate that vWF-platelet interactions can occur in the absence of artificial agonists or chemical modification of vWF. They suggest a possible mechanism for platelet aggregation in stenosed or partially obstructed arterial vessels in which the platelets are subjected to relatively high levels of shear stress.     

Acquired von Willebrand disease and storage pool disease in chronic myelocytic leukemia

Platelet function was evaluated in 20 patients with chronic myelocytic leukemia (CML), all Ph positive. Seven showed abnormal epinephrine-induced aggregation, while four had impaired both ADP- and collagen-induced aggregation. The platelets of all patients aggregated with arachidonic acid, thus ruling out cyclooxygenase or lipoxygenase deficiency. The intracellular concentrations of ATP and ADP were significantly below normal, and the ratio of ATP/ADP was greater than normal in all 12 patients. ATP released from platelets by Lumi-aggregometer was reduced. In four patients with abnormal ristocetin-induced aggregation, vWF:Ag, RCoF, and FVIII:C were all reduced. No significant inactivation of factor VIII was induced in normal plasma by incubation with patient's plasma. The crossed immunoelectrophoretic analysis revealed that vWF:Ag in these patients was mainly composed of more anodic component as compared with that of normal plasma. The ratio of vWF:Ag/RCoF was significantly greater than normal. A marked increase of factor VIII and a rapid return of vWF:Ag and RCoF to the baseline after the 1-deamino-8-arginine vasopressin (DDAVP) infusion were observed. Transient increase in vWF:Ag after the infusion of DDAVP appeared with less anodic forms and in the same relative proportion as that in normal plasma. The present study shows that in some patients with CML storage pool disease occurs with acquired von Willebrand disease.     

Increased fragmentation of von Willebrand factor, due to abnormal cleavage of the subunit, parallels disease activity in recurrent hemolytic uremic syndrome and thrombotic thrombocytopenic purpura and discloses predisposition in families. The Italian Registry of Familial and Recurrent HUS/TTP.

We investigated here the changes in von Willebrand factor (vWF) multimers in recurrent, sporadic and familial forms of hemolytic uremic syndrome (HUS)/thrombotic thrombocytopenic purpura (TTP) to see whether they are actually proteolyzed in vivo in these patients. Molecular determinants of fragments in vWF were also characterized to identify possible sites of cleavage of the subunit. Unusually large vWF multimers were found in blood of 8 of 10 patients with recurrent HUS/TTP, both in the acute phase and in remission, but never in familial and sporadic cases. Instead, all of the groups showed evidence of enhanced fragmentation of vWF multimers during the acute phase. Increased fragmentation was also shown by decrease in native 225-kD vWF subunit. In recurrent and sporadic HUS/TTP, enhanced fragmentation normalized at remission, but the abnormality persisted in familial HUS/TTP patients. The latter findings suggest that patients with familial HUS/TTP may have a congenital abnormality in vWF processing. Analysis with specific monoclonal antibodies showed the presence of the normal vWF fragments with apparent molecular mass of 189, 176, and 140 kD in all patients; however, in 6 recurrent and in 5 familial cases, novel fragments that differed in size from normal ones were found. The size of these abnormal fragments differed from one patient to another and none of them was ever found in normal plasma. These results documented, for the first time in HUS/TTP, an abnormal cleavage of the vWF subunit that might account for the increased fragmentation observed in these patients.     

Platelet – von Willebrand factor interactions in Type IIB von Willebrand's disease

Type IIB von Willebrand's disease (vWD) is a distinct form of this disorder in which the largest multimers of the von Willebrand factor (vWF) are lacking in plasma but present in platelets. When the vasopressin analogue, l-deamino-8-D-arginine vasopressin (DDAVP), is given to patients with type IIB vWD, an abnormal vWF is released to plasma. This vWF causes thrombocytopenia in vivo and platelet aggregation in vitro. Aggregation occurs in the plasma milieu and thus at physiological fibrinogen concentration. In this study we demonstrate that IIB post-DDAVP vWF aggregated only metabolically active platelets. The platelet aggregation was completely inhibited by EDTA and PGE₁, and either inhibited or greatly weakened by ASA, demonstrating the role of divalent cations and thromboxane A₂ formation. In spite of inhibiting platelet aggregation, EDTA, PGE₁ and ASA did not prevent platelet binding of IIB post-DDAVP vWF. An antiserum against GP Ib made normal platelets less responsive to the IIB vWF although neither platelet aggregation nor vWF binding were completely prevented. The aggregation was fibrinogen-dependent and platelets from patients with Glanzmann's thrombasthenia were unresponsive. The studies provide evidence that IIB post-DDAVP vWF is bound to unstimulated platelets and that the interaction between vWF and platelets in type IIB vWD is different from ristocetin-induced as well as thrombin- and epinephrine-induced binding to platelets of normal vWF.     

Spontaneous platelet aggregation during pregnancy in a patient with von Willebrand disease type IIB can be blocked by monoclonal antibodies to both platelet glycoproteins Ib and IIb/IIIa

Spontaneous platelet aggregation appeared in a patient with von Willebrand disease type IIB during the 37th week of pregnancy. This phenomenon was not associated with symptoms of thrombosis and the patient delivered by caesarean section with no complications. Her platelet-poor plasma (PPP) aggregated normal platelet-rich plasma (PRP) and washed platelets. Aggregation was inhibited by monoclonal antibodies with known specificity for the platelet receptors of von Willebrand factor (vWF), i.e. the glycoprotein Ib (GPIb) and the GPIIb/IIIa complex. A monoclonal antibody, which selectively inhibits the binding of vWF to the GPIIb/IIIa complex, did not block aggregation, suggesting that spontaneous aggregation is not dependent on the binding to GPIIb/IIIa of vWF from patient plasma. Aggregation induced by patient plasma could also be blocked either by two monoclonal antibodies raised against vWF or by a fragment derived from trypsin digestion of normal vWF which blocks the ristocetin-induced binding of normal vWF to platelets. These findings indicate that the spontaneous platelet aggregation in this patient results from the binding of her vWF to GPIb but is independent from the binding of her vWF to GPIIb/IIIa.     

A new congenital platelet abnormality characterized by spontaneous platelet aggregation, enhanced von Willebrand factor platelet interaction, and the presence of all von Willebrand factor multimers in plasma

A case is reported of a 49-year-old woman with a mild bleeding tendency. Her bleeding time, platelet count and size, plasma ristocetin cofactor activity, von Willebrand factor (vWF) antigen, and vWF multimeric pattern are all within normal limits. Spontaneous platelet aggregation is observed when citrated platelet-rich plasma (PRP) is stirred in an aggregometer cuvette. This aggregation is completely is only slightly diminished by an antiglycoprotein (GP) IIb/IIIa or by an anti GPIb monoclonal antibody. The patient's PRP shows increased sensitivity to ristocetin. The distinct feature of this patient, also present in two family members studied, is that platelet aggregation is initiated by purified vWF in the absence of any other agonist. The vWF- induced platelet aggregation is abolished by anti-GPIb and anti- GPIIb/IIIa monoclonal antibodies and by EDTA (5 mmol/L). Apyrase inhibits the second wave of aggregation. Patient's platelets in PRP are four to six times more reactive to asialo vWF-induced platelet aggregation than normal platelets. The amount of radiolabeled vWF bound to platelets in the presence of either low concentration of ristocetin or asialo vWF was increased 30% compared with normal. The patient's platelet GPIb was analyzed by SDS page and immunoblotting and by binding studies with anti-GPIb monoclonal antibodies showed one band with slightly increased migration pattern and a normal number of GPIb molecules. Unlike the previously reported patients with pseudo or platelet-type von Willebrand disease, this patient has normal vWF parameters.     

Botrocetin- and polybrene-induced platelet aggregation in platelet-type von Willebrand disease

It has been reported that botrocetin, a Bothrops venom factor, induces platelet aggregation dependent on von Willebrand factor (vWF), and that platelet aggregation induced by Polybrene, a synthetic polycation, is enhanced by vWF. This report describes the platelet aggregability on stimulation with botrocetin and Polybrene in four patients with platelet-type von Willebrand disease (vWD) who showed increased platelet aggregation with low concentrations of ristocetin as the result of a platelet abnormality. Enhanced platelet aggregability with botrocetin was observed in platelet-rich plasma (PRP) from the patients. Platelet aggregation induced by botrocetin in a mixture of normal washed platelets and patient plasma was either decreased or normal, being dependent on the amount of plasma vWF. In contrast with ristocetin and botrocetin, Polybrene did not cause increased aggregation of patient PRP. Polybrene aggregated normal washed platelets less extensively in the presence of patient plasma than normal plasma. These studies demonstrated that botrocetin induced heightened interaction between platelets and vWF, but Polybrene did not, in platelet-type vWD, and that the enhanced responsiveness of patient platelets to botrocetin is related to an intrinsic platelet abnormality.     

Calpain proteolysis of von Willebrand factor enhances its binding to platelet membrane glycoprotein IIb/IIIa: an explanation for platelet aggregation in thrombotic thrombocytopenic purpura

We have previously observed calpain activity (calcium-dependent cysteine protease) in sera from patients with acute thrombotic thrombocytopenic purpura (TTP). The calpain activity was not present following recovery and was not detected in other thrombocytopenic disorders. We postulated that this enzyme could participate in the pathogenesis of TTP. Because other investigators have demonstrated abnormalities of von Willebrand factor (vWF) in patients with TTP, we proposed that calpain might interact with vWF in TTP. To challenge this hypothesis, we measured the binding of untreated and calpain-treated vWF to normal and ADP or calpain activated platelets. Untreated vWF bound in a specific and saturable fashion to activated platelets, but only at low (30 microM) calcium concentrations. Von Willebrand factor did not bind to activated platelets at physiological (2 mM) calcium concentrations. Calpain proteolysis of vWF changed the binding characteristics of the vWF so that it had greatly increased binding to both ADP and calpain activated platelets. The calpain-proteolyzed vWF bound to activated platelets at both low and physiological calcium concentrations, and was capable of causing platelet aggregation. The calpain-proteolyzed vWF bound to the activated platelets via glycoproteins IIb/IIIa as demonstrated by inhibition studies using monoclonal antibodies against glycoproteins IIb/IIIa and Ib. It also had a high binding affinity and was capable of inhibiting the binding of radiolabelled fibrinogen to the activated platelets at physiological calcium concentrations. Calpain also proteolyzed fibrinogen, but the calpain altered fibrinogen had normal platelet reactivity. These studies provide further insight into the pathogenesis of the platelet aggregation of thrombotic thrombocytopenic purpura. Calpain proteolyses vWF and can produce the characteristic loss of large multimers seen on sodium dodecyl sulphate (SDS)-agarose gel electrophoresis. The altered vWF is highly reactive with activated platelets and binds to platelet glycoproteins IIb/IIIa and participates in formation of the platelet aggregates that characterize this disease.     

von Willebrand factor synthesized by endothelial cells from a patient with type IIB von Willebrand disease supports platelet adhesion normally but has an increased affinity for platelets.

Endothelial cells were isolated from the umbilical vein of a patient with subtype IIB von Willebrand disease, and the biosynthesis and function of von Willebrand factor (vWF) synthesized by these cells were compared with those of vWF synthesized by endothelial cells from normal individuals. The patient's endothelial cells synthesized, stored, and secreted vWF indistinguishably from normal endothelial cells: it was synthesized as a prepolypeptide of Mr 270,000 and had a mature form of Mr 220,000; the full spectrum of multimers was found both inside the cells and in the culture medium; it was stored normally, in the Weibel-Palade bodies; and similar amounts of vWF were secreted into the medium and deposited in the extracellular matrix. In a perfusion set-up, the extracellular matrix from IIB cells supported platelet adhesion similarly to the matrix from normal cells. vWF secreted constitutively by IIB cells into the culture medium bound to platelets at concentrations of ristocetin lower than those necessary for vWF from normal cells. vWF stored in the Weibel-Palade bodies of type IIB cells was released upon stimulation with phorbol ester and bound almost completely to platelets even in the absence of ristocetin. Moreover, spontaneous platelet aggregation was induced by vWF synthesized by type IIB cells. These data support the hypothesis that the absence of highly multimeric forms of vWF in plasma of type IIB von Willebrand disease patients is due to specific removal of these multimers by platelets.     

Von Willebrand factor can support platelet aggregation via interaction with activated GPIIb-IIIa and GPIb

In this study we investigated mechanisms of platelet interaction with von Willebrand factor (vWF) induced by activating anti-glycoprotein (GP)IIb-IIIa antibody CRC54 directed against LIBS (ligand-induced binding site epitope) in GPIIIa. It was demonstrated that aggregation of washed platelets (measured in Born aggregometer) could be stimulated by CRC54 not only in the presence of fibrinogen but vWF as well. The level of aggregation induced in the presence of saturating concentrations of vWF (approximately 80 microg/ml) was even higher than that in the presence of 1 mg/ml of fibrinogen. Aggregation supported by vWF unlike fibrinogen supported aggregation was almost completely inhibited not only by GPIIb-IIIa antagonists (F(ab')2 fragment of blocking anti-GPIIb-IIIa antibody CRC64 and peptidomimetic aggrastat) but also by anti-GPIb blocking antibody AK2. Aggregation response induced by CRC54 in the presence of vWF was much lower when normal platelets were substituted with GPIb-deficient platelets and this residual aggregation was not affected by anti-GPIb antibody AK2 but still inhibited by anti-GPIIb-IIIa blocking antibody fragment. CRC54-induced aggregation supported by vWF (as well as by fibrinogen) was only partially inhibited by prostaglandin E1, indicating that at least its initiation does not require activation of platelets. CRC54, both in the presence of vWF and fibrinogen, failed to stimulate serotonin secretion at physiological Ca2+ concentration of 1 mM, although substantial release reaction was detected when Ca2+ concentration was decreased to 0.1 mM. CRC54 could also stimulate platelet interaction with immobilized vWF and fibrinogen. However, unlike platelet aggregation in suspension mediated by flow phase vWF, platelet adhesion to adsorbed vWF (in a same way as to fibrinogen) was inhibited only by GPIIb-IIIa but not GPIb antagonists. The data obtained indicated that vWF support platelet aggregation induced by activating anti-GPIIb-IIIa via interaction with two receptors - activated GPIIb-IIIa and GPIb.     

Mechanisms of platelet aggregation by Streptococcus sanguis, a causative organism in infective endocarditis

Summary. The ability of certain strains of Streptococcus sanguis to aggregate human platelets in vitro may be related to their virulence in the pathogenesis of infective endocarditis. We have studied the mechanisms of aggregation of human platelets by S. sanguis strain NCTC 7863. Platelet aggregation follows incubation of S. sanguis cells with platelet-rich plasma from normal, healthy adults, after a lag of 7–19 min. Platelet aggregation was accompanied by 5-hydroxytryptamine release and thromboxane B₂ production. Aggregation was prevented by aspirin and by EDTA. Platelets from two patients with Glanzmann's thrombasthenia did not respond to bacteria. Fixed, washed platelets resuspended in normal plasma were not agglutinated by S. sanguis.
Blocking the glycoprotein Ib receptor with a monoclonal antibody inhibited aggregation of PRP. However, S. sanguis did not induce von Willebrand factor (vWF) binding to platelets; nor did the bacteria prevent ristocetin-induced platelet agglutination or vWF binding. The aggregation response was not related to plasma vWF activity levels in normal subjects or in patients with von Willebrand's disease.
The platelet response to S. sanguis therefore resembles true aggregation, requiring the cyclo-oxygenase pathway and the presence of glycoprotein IIb/IIIa. The mechanism also involves glycoprotein Ib, but not apparently through irreversible binding of vWF.     

Specific von Willebrand factor-cleaving protease in thrombotic microangiopathies: a study of 111 cases

Retrospective studies of patients with thrombotic microangiopathies (TMAs) have shown that a deficient activity of von Willebrand factor (vWF)-cleaving protease is involved in thrombotic thrombocytopenic purpura (TTP) but not in the hemolytic-uremic syndrome (HUS). To further analyze the relevance of this enzymatic activity in TMA diagnosis, a 20-month multicenter study of vWF-cleaving protease activity was conducted in adult patients prospectively enrolled in the acute phase of TMA. Patients with sporadic (n = 85), intermittent (n = 21), or familial recurrent (n = 5) forms of TMA (66 manifesting as TTP and 45 as HUS) were included. TMA was either idiopathic (n = 42) or secondary to an identified clinical context (n = 69). vWF-cleaving protease activity was normal in 46 cases (7 TTP and 39 HUS) and decreased in 65 cases (59 TTP and 6 HUS). A protease inhibitor was detected in 31 cases and was observed only in patients manifesting TTP with a total absence of protease activity. Among the 111 patients, mean vWF antigen levels were increased and the multimeric distribution of vWF was very heterogeneous, showing either a defect of the high-molecular-weight forms (n = 40), a normal pattern (n = 21), or the presence of unusually large multimers (n = 50). Statistical analysis showed that vWF-protease deficiency was associated with the severity of thrombocytopenia (P <.01). This study emphasizes that vWF-cleaving protease deficiency specifically concerns a subgroup of TMA corresponding to the TTP entity.