Laurie P. McKeown

Activated platelets in paroxysmal nocturnal haemoglobinuria

Summary. One of the major causes of morbidity and mortality in paroxysmal nocturnal haemoglobinuria (PNH) is venous thrombosis. We have studied fibrinolysis, coagulation and platelets in 11 patients with PNH in an attempt to identify the possible mechanism(s) of thrombosis in PNH. In this study we did not identify any fibrinolytic defects, evidence of coagulation activation, nor reduction in coagulation inhibitors. In contrast, in this cohort of 11 PNH patients we have identified varying degrees of platelet activation as defined by the surface expression of activation dependent proteins and the binding of adhesive proteins to the platelet surface. The thrombotic events in PNH usually occur in the venous system. Our studies and previous experimental studies suggest that anti‐platelet therapy may be efficacious in reducing the incidence and severity of venous thrombosis in PNH.

Platelet von Willebrand factor: Comparison with plasma von Willebrand factor

Platelet von Willebrand factor (vWf) was compared to its plasma counterpart. The platelet vWf was different from plasma vWf in that the multimeric organization was different, larger multimers were present, and the ratio of vWf activity to antigen was higher. Platelet and plasma vWf were similar in their antigenic reactivity in the electroimmunoassay and by liquid phase radioimmunoassay. The amount of vWf activity in large platelets was significantly higher than in normal platelets while the antigen content, although somewhat higher, was not significant. These studies show differences between normal platelet and plasma vWf, and also suggest that platelet size must be considered when platelet vWf is measured in disease states.

1-desamino-8-arginine-vasopressin corrects the hemostatic defects in type 2B von Willebrand's disease

DDAVP is effective treatment in most types of von Willebrands disease; however, in type 2B von Willebrands disease the use of DDAVP has been contraindicated due to DDAVP‐induced thrombocytopenia. Several reports have confirmed the thrombocytopenic effects of DDAVP and the presence of circulating platelet aggregates in type 2B von Willebrands disease. We have infused three type 2B patients with DDAVP. The three patients had different mutations of their vWf. All three patients had a missense mutation which resulted in a single amino acid substitution in the disulfide loop of the A1 domain. Administration of 20 μg of DDAVP resulted in significant elevations of factor VIII, vWf antigen, and ristocetin cofactor levels. In contrast to other studies, DDAVP did not induce or enhance thrombocytopenia in these three patients. When blood was obtained by fingerstick and diluted into sodium oxalate (Unopette®) or EDTA (Microvette®), the platelet counts did not change over 4 hr. In contrast, blood collected directly into evacuated tubes containing sodium citrate, lithium heparin, or EDTA consistently demonstrated varying degrees of thrombocytopenia and platelet clumping. We also observed a shortening of the preinfusion bleeding time over the 4 hr period. All three patients have been studied twice and each has shown consistent results. DDAVP appears to be a useful form of treatment in type 2B vWd. (This article is a US Government work and, as such, is in the public domain in the United States of America.)

Purification and characterization of human platelet von Willebrand factor

Summary. Platelet von Willebrand factor (vWf) was purified from human platelet concentrates. The multimeric structure of the purified platelet vWf was similar to that observed in the initial platelet lysate, and, like the platelet lysate, the purified platelet vWf contained higher molecular weight multimers than plasma vWf. The apparent molecular weight of the reduced platelet vWf subunit was similar to the plasma vWf subunit. The N‐terminal amino acid of the purified platelet and plasma vWf was blocked. In concentration dependent binding to botrocetin‐ or ristocetin‐stimulated platelets, 125I‐plasma vWf bound with a higher affinity than platelet. The ristocetin cofactor activity per mg of purified plasma vWf was 5‐fold greater than the platelet vWf activity. Platelet and plasma vWf bound to collagen with similar affinities; however, platelet vWf bound to thrombin‐stimulated platelets and to heparin with a higher affinity than plasma vWf. The differences in the binding affinity(s) of plasma and platelet vWf to platelet GPIb and GPIIb/IIIa and extracellular matrix proteins may reflect different roles for plasma and platelet vWf in the initial stages of haemostasis and thrombosis.

Platelet von Willebrand Factor

von Willebrand factor (vWF) circulates in the blood in two distinct compartments. One, plasma vWF, is synthesized and released from endothelial cells; the second, synthesized by megakaryocytes, circulates in platelets primarily stored in the alpha granules. Recent experimental and clinical studies of von Willebrands disease (vWD) indicate that platelet vWF plays an important role in the bleeding time determination and the degree of clinical bleeding in vWD. Platelet vWF is released from the platelet alpha granules by various agonists and then rebinds to the glycoprotein IIb/IIIa complex. Fibrinogen or monoclonal antibodies against this complex inhibit 60 to 70% of the expression of platelet vWF. Aspirin inhibits 80% of the adenosine diphosphate-induced platelet vWF surface expression, and the platelet vWF surface expression that is not inhibited by aspirin can be almost totally inhibited by disruption of the platelet cytoskeleton. Platelet vWF may, in part, be expressed in the open canalicular system prebound to a receptor. Transfusion studies have shown that correction of the bleeding time in severe vWD requires both plasma and platelet vWF. On the basis of numerous studies, we hypothesize that platelet vWF plays an important role in platelet interaction with the subendothelial surfaces under conditions of high shear stress. After platelet contact, platelet vWF is released, binds to the glycoprotein IIb/IIIa complex, and forms a bridge between the subendothelial surface and the platelet, which initiates and supports platelet spreading. Platelet vWF also acts as an intercellular bridge between platelets and thereby promotes platelet aggregation. This process is important not only in the initial steps of hemostasis but also in the process of thrombosis.

Platelet adhesion at high shear rates: The roles of von Willebrand factor/GPIb and the β1 integrin α2β1

We have previously described a monomeric rvWf fragment, Leu504-Lys728 that contains one disulfide bond linking Cys509-Cys695. This fragment, VCL, has previously been shown to inhibit vWf-ristocetin, asialo-vWf, and botrocetin-induced vWf binding and aggregation of platelets. VCL inhibited 50% of vWf binding to heparin, but it did not inhibit vWf binding to type I collagen. At a high shear force (2600-1 sec), VCL inhibited platelet adhesion to the subendothelial surface of human umbilical arteries. The maximum inhibition of platelet adhesion was 83 +/- 4% at a VCL concentration of 7.6 mumol/L. Various monoclonal anti-Very Late Activation antigens (VLA) antibodies were added to the VCL and tested for their ability to enhance the inhibition of platelet adhesion at high shear forces. Of all of the VLA antibodies tested, only the anti-VLA-2 antibody (176D7) inhibited platelet aggregation in the absence of VCL and enhanced the inhibition of platelet adhesion in the presence of VCL. The VLA-2 antibody and VCL together inhibited 96 +/- 4% of platelet adhesion at high shear forces.

Platelet aggregation induced by type IIb platelet von Willebrand factor

Summary Platelet lysates from five patients with a form of type IIb von Willebrands disease (vWd), associated with spontaneous platelet aggregation and thrombocytopenia, induced platelet aggregation of normal and other vWds platelet‐rich plasma (PRP). Platelet lysate from normals, type I or type IIa vWd did not cause platelet aggregation of normal PRP. When polyclonal monospecific antibodies directed against plasma von Willebrand factor (vWf) were incubated with the type IIb platelet lysate, they inhibited the platelet aggregation. Monoclonal antibodies directed against the glycoprotein (GP) Ib binding domain of plasma vWf incubated with the type IIb platelet lysate did not inhibit the platelet aggregation. Normal platelets suspended in afibrinogenaemic plasma did not aggregate when type IIb vWd platelet lysate was added. Normal platelets incubated with monoclonal antibodies directed against the fibrinogen and vWf binding site(s) on the GPIIb/IIIa were not aggregated by the type IIb platelet lysate. Bernard‐Soulier PRP aggregated when type IIb vWd platelet lysate was added, while Glanzmanns thrombasthenic platelets did not. Peptides containing the RGDS sequence or the sequence of the carboxy terminal 15 amino acids of the gamma chain of fibrinogen inhibited the type IIb vWd platelet lysate‐induced platelet aggregation. These data suggest that type IIb platelet vWf can cause platelet aggregation of PRP without the addition of any agonist. This interaction is different from that observed with the plasma vWf from these patients.

Platelet von Willebrand factor in Hermansky-Pudlak syndrome

The Hermansky‐Pudlak Syndrome (HPS) is an autosomal recessive inherited disorder characterized by oculocutaneous albinism, tissue accumulation of ceroid pigment, and a mild to moderate bleeding diathesis attributed to storage‐pool deficient (SPD) platlets. Patients have platelet aggregation and release abnormalities. In addition, low levels of plasma von Willebrand factor (vWF) antigen in some HPS patients have been associated with a greater bleeding tendency than would be predicted from either condition alone. Other HPS patients have severe bleeding despite normal levels of plasma vWF, suggesting that at least one additional factor is responsible for their bleeding diathesis. Because platelet vWF levels have been well correlated with clinical bleeding times in patients with von Willebrands disease, we have measured the platelet vWF activity and antigen levels in 30 HPS patients and have attempted to correlate their clinical bleeding with these values. The platelet vWF activity levels in patients was significantly lower than that of normal subjects (P < 0.0001). The patients as a group also had slightly lower values of plasma vWF activity when compared with normals (P ∼ 0.03). In 11 of the HPS patients, the multimeric structure of plasma vWF showed a decrease in the high molecular weight multimers and an increase in the low molecular weight multimers. In correlating the platelet and plasma vWF values with the bleeding histories, we were not able to show a predictable relationship in the majority of the patients. Am. J. Hematol. 59:115–120, 1998.

Effect of platelet count on the DDAVP-induced shortening of the bleeding time in thrombocytopenic Gaucher's patients

As part of an investigative protocol requiring serial liver biopsies in patients with Type I Gauchers disease who were receiving enzyme replacement therapy, we gave 11 patients a total of 15 infusions of DDAVP (0.3 micrograms/kg IV) and measured the Simplate bleeding time pre- and postin-fusion. All patients were thrombocytopenic (one patient 100-150,000; seven 66-99,000; and three 50-65,000/microliters). Nine of 15 infusions resulted in at least a 2-min shortening of the bleeding time; in 6/11 infusions in which the platelet count was greater than 65,000, the bleeding time shortened to less than or equal to 10 min, in 1/11 it shortened greater than or equal to 2 min but not to less than or equal to 10 min. In only 2/4 infusions given to patients with platelet counts less than 65,000 did the bleeding time demonstrate any significant shortening. None of the six liver biopsies performed in the patients whose bleeding time shortened to less than or equal to 10 min resulted in any significant bleeding; blood products were not transfused either pre- or postprocedure. These limited data demonstrate that a high percentage of patients with platelet counts of 65-100,000/microliters can manifest a significant shortening of bleeding time following a standard infusion of DDAVP, and that in approximately half of these patients the bleeding time will shorten enough to safely allow the performance of a liver biopsy without the need for prophylactic transfusion of blood products.

Platelet activation and alpha granule secretion in type IIB von Willebrand's disease

Type IIB von Willebrand disease is characterized by enhanced ristocetin‐induced platelet aggregation, spontaneous platelet aggregation, thrombocytopenia and the absence of the largest plasma von Willebrand factor (vWf) multimers. The absence of the largest plasma vWf multimers is related to their enhanced binding to platelets. The abnormal affinity of the IIB von Willebrand factor to platelets results in thrombocytopenia, but the mechanism is not known. We have studied the platelets from three patients with type IIB von Willebrand disease and have found evidence of platelet activation and alpha granule secretion as defined by increased amounts of von Willebrand factor, fibrinogen and the alpha granule protein PADGEM/GMP‐140 on the surface of these platelets. The degree of thrombocytopenia appears to be directly related to the number of platelets with fibrinogen bound to the surface. PADGEM/ GMP‐140, an alpha granule membrane protein, fuses with the platelet plasma membrane after activation and is a site on platelets which binds to neutrophils or monocytes. This alpha granule protein may play an additional role in platelet clearance and thrombocytopenia in type IIB von Willebrand disease. This may, in part, explain the absence of thromboembolic phenomena despite the presence of activated platelets in patients with type IIB von Willebrand disease.