Effects of monoclonal antibodies against the platelet glycoprotein IIb/IIIa complex on thrombosis and hemostasis in the baboon.

To assess the hemostatic consequences and antithrombotic effectiveness of blocking the platelet glycoprotein (GP) IIb/IIIa receptor for fibrinogen and other adhesive glycoproteins in vivo, well characterized murine monoclonal antibodies against the platelet GP IIb/IIIa complex, AP-2 and LJ-CP8, were infused intravenously into baboons. Four animals each received doses of 0.2, 0.4, and 1.0 mg/kg of purified AP-2 IgG, and three animals were given 1.0 mg/kg of the F(ab)2 fragment of AP-2. Five additional animals were given 10 mg/kg LJ-CP8 IgG. At the highest dose, radiolabeled AP-2 IgG bound to an average of 33,000 sites on the circulating platelets. Serial measurements included platelet count, bleeding time, platelet aggregation (induced by ADP, collagen, and gamma-thrombin), and 111In-platelet deposition onto Dacron vascular grafts. Bleeding times were markedly prolonged after injection of 1.0 mg/kg AP-2 IgG (19.2 +/- 3.4 min), 1.0 mg/kg AP-2 F(ab)2 (16.5 +/- 1.8 min), and 10 mg/kg LJ-CP8 (greater than 30 min) vs. control studies (4.6 +/- 0.2 min), and remained prolonged for 48 h. With each antibody platelet aggregation was initially reduced or absent, with partial recovery over 48 h in a manner that was inversely related to dose. AP-2, both whole IgG and F(ab)2 fragment, but not LJ-CP8, caused a dose-dependent reduction (20-46%) in the circulating platelet count over 24 h. Neither AP-2 nor LJ-CP8 caused a reduction in intraplatelet platelet factor 4, beta-thromboglobulin, or [14C]serotonin. Graft-associated platelet thrombus formation was reduced by 73% (1.0 mg/kg AP-2 IgG and 10 mg/kg LJ-CP8) and 53% (1.0 mg/kg AP-2 F(ab)2) relative to control values. In contrast, neither heparin (100 U/kg) nor aspirin (32.5 mg/kg twice a day) showed antithrombotic efficacy in this model. Thus, antibodies that functionally alter the platelet GP IIb/IIIa complex may produce immediate, potent, and transient, antihemostatic, and antithrombotic effects.

Proteolysis of von Willebrand factor and shear stress-induced platelet aggregation in patients with aortic valve stenosis.

BACKGROUND: Excessive bleeding may complicate congenital cardiac defects. To explain the pathogenesis of this abnormality, we evaluated selected parameters of primary hemostasis in patients with aortic valve stenosis before and after corrective surgery. METHODS AND RESULTS: We examined shear-induced platelet aggregation with the filter aggregometer test and von Willebrand factor (vWF) structure by evaluating the multimeric distribution and extent of subunit proteolysis. The platelet count was reduced before corrective surgery, and shear-induced platelet aggregation was impaired. Moreover, vWF multimers of higher molecular mass were decreased, and proteolytic subunit fragments were increased. After correction of the cardiac defect, all of these parameters returned to normal. CONCLUSIONS: Alterations of vWF and platelet function may contribute to the bleeding diathesis in patients with aortic valve stenosis. Improvement after corrective surgery suggests that the passage of blood through a stenosed aortic valve may result in shear forces that induce vWF interaction with platelets in the circulation and, in turn, trigger platelet clearance, vWF degradation, and the impairment of primary hemostasis.

Acquired dysfunction due to the circulation of "exhausted" platelets.

An acquired platelet functional defect was found to be present in eight patients who presented with various clinical conditions--three with renal allograft rejection, three with the hemolytic uremic syndrome or thrombotic thrombocytopenic purpura, one with acute consumption coagulopathy due to an incompatible transfusion and one with systemic lupus erythematosus. They showed defective platelet aggregation and reduced levels of adenine nucleotides and serotonin with abnormal uptake and storage of the amine. The bleeding time was more prolonged than predicted from the platelet count. These abnormalities were strikingly similar to those occurring in patients with congenital storage pool deficiency. The acquired defect is thought to be related to the presence in the circulation of "exhausted" platelets following their in vivo exposure to inducers of the release reaction such as damaged endothelium, thrombin and immune complexes. The bleeding tendency of the underlying diseases might be aggravated by the impairment of platelet function.

Detection of circulating released platelets after renal transplantation.

In recipients of renal transplants, the biochemical defect(s) that underlies increased deposition of platelets in the graft and their shortened survival in the circulation are poorly understood. Forty-six recipients of kidney allografts, with and without rejection signs (13 acute rejections (ARs), 15 chronic rejections (CRs), and 18 functioning transplants (FTs), had lower platelet serotonin (5HT) and higher plasma beta-thromboglobulin than normal controls (NCs). These abnormalities were more pronounced in patients with ARs than with CRs but were also present in patients with FTs. All groups of transplant recipients showed an abnormal metabolism of platelet arachidonate, as expressed by low serum levels of thromboxane B2. In AR, plasma fibrinopeptide A (FPA) was significantly high whereas FPA levels were unchanged in CR and in FT. These findings suggest that in patients with renal transplants, the platelet release reaction has occurred in vivo, resulting in the secretion of granule-bound substances and the circulation of partially empty platelets. Vasoactive, mitogenic, and proaggregatory substances released from platelets might damage the graft and aggravate the rejection process.

Effect on platelet functions of derivatives of cyclic nucleotides.

Derivatives of cyclic nucleotides were evaluated for their ability to inhibit platelet aggregation and the release reaction. Derivatives substituted in position 8 (mainly 8-Br-cyclic GMP) were more active than 3'-5' cyclic AMP, and their relative potency in inhibiting platelet aggregation and 14C-serotonin release was comparable to that of N62-0'-dibutyryl-cyclic AMP. Compounds substituted in position 6 or 2'-0 were not effective. The active compounds, which were also tested for their ability to stimulate platelet adenylate cyclase or to inhibit cyclic AMP phosphodiesterase, did not modify the intracellular levels of cyclic AMP. Since previous animal experiments have shown that these derivatives cause less side effects than cyclic AMP and its dibutyryl derivative in animals, it is suggested that modification of the cyclophosphate molecule might make it possible to find compounds active only on platelet function without interfering with other biological systems.

Biochemical and metabolic aspects of platelet dysfunction in chronic myeloproliferative disorders.

Fifty-two patients with chronic myeloproliferative disorders (13 with polycythemia vera; 23 with primary thrombocythemia; 6 with myelofibrosis and 10 with chronic granulocytic leukemia) had low platelet levels of adenine nucleotides and serotonin and abnormal uptake and storage of the amine. The storage pool deficiency was confined to the substances contained in the platelet dense bodies, because alpha-granule and lysosome markers were present in normal amounts. In chronic granulocytic leukemia the storage defect was usually less marked but was accompanied by a decreased formation of thromboxane B2 and normal platelet aggregation in response to arachidonic acid. There was no clearcut relationship of these biochemical abnormalities to prolongation of bleeding time or to thrombotic and hemorrhagic symptoms. The defect was still present in 15 patients after treatment had returned the cell counts to the normal range. Normal levels of 5HT and adenine nucleotides were observed in 8 patients whose platelet counts were high after splenectomy for non-hematological reasons. These findings suggest that biochemical abnormalities are related to the presence in the bone marrow of abnormal clones, resulting in the production of defective platelets.

Porcine von Willebrand factor binding to human platelet GPIb induces transmembrane calcium influx.

Porcine von Willebrand factor (P-vWF) binds to human platelet glycoprotein (GP) Ib and, upon stirring (1500 rpm/min) at 37 degrees C, induces, in a dose-dependent manner, a transmembrane flux of Ca2+ ions and platelet aggregation with an increase in their intracellular concentration. The inhibition of P-vWF binding to GP Ib, obtained with anti GP Ib monoclonal antibody (LJ-Ib1), inhibits the increase of intracellular Ca2+ concentration ([Ca2+]i) and platelet aggregation. This effect is not observed with LJ-Ib10, an anti GP Ib monoclonal antibody which does not inhibit the vWF binding to GP Ib. An anti GP IIb-IIIa monoclonal antibody (LJ-CP8) shown to inhibit the binding of both vWF and fibrinogen to the GP IIb-IIIa complex, had only a slight effect on the [Ca2+]i rise elicited by the addition of P-vWF. No inhibition was also observed with a different anti GP IIb-IIIa monoclonal antibody (LJ-P5), shown to block the binding of vWF and not that of fibrinogen to the GP IIb-IIIa complex. PGE1, apyrase and indomethacin show a minimal effect on [Ca2+]i rise, while EGTA completely blocks it. The GP Ib occupancy by recombinant vWF fragment rvWF445-733 completely inhibits the increase of [Ca2+]i and large aggregates formation. Our results suggest that, in analogy to what is seen with human vWF under high shear stress, the binding of P-vWF to platelet GP Ib, at low shear stress and through the formation of aggregates of an appropriate size, induces a transmembrane flux of Ca2+, independently from platelet cyclooxygenase metabolism, perhaps through a receptor dependent calcium channel. The increase in [Ca2+]i may act as an intracellular message and cause the activation of the GP IIb-IIIa complex.

Evaluation of the abnormal platelet function in von Willebrand disease by the blood filtration test.

We have evaluated platelet function in different subtypes of von Willebrand disease (vWD) by pushing blood through the capillary-sized channels of a glass filter. Patients, including those with type IIB vWD, showed lower than normal platelet retention and increased cumulative number of blood drops passing through the filter as a function of time. In contrast, shear-induced platelet aggregation, measured in the cone-and-plate viscometer, was paradoxically increased in type IIB patients. Treatment with 1-desamino-8-D-arginine vasopressin (DDAVP) tended to normalize the filter test in patients with type I-platelet normal and type I-platelet low vWD, but infusion of a factor VIII/von Willebrand factor (vWF) concentrate lacking the largest vWF multimers was without effect in type 3 patients. Experiments with specific monoclonal antibodies demonstrated that the A1 and A3 domains of vWF, as well as the glycoproteins Ib alpha and IIb-IIIa on platelets, are required for platelet retention in the filter. Thus, the test may reflect vWF function with regard to both platelet adhesion and aggregation under high shear stress, and provide relevant information on mechanisms involved in primary hemostasis.

Asymptomatic liver disease in haemophiliacs.

The incidence of jaundice and of abnormal liver function tests has been assessed in 91 multitransfused patients with severe haemophilia A and B. Tests of hepatocyte function were within the normal range in the majority of patients. On the contrary, tests of biliary cell function, liver cell damage, and bromsulphthalein retention gave high rates of abnormal values, which tended to increase with age. Hepatitis B surface antigen was present in 8% and the corresponding antibody in 66% of the cases; 18% had a history of jaundice. All patients were asymptomatic and only a minority showed clinical signs of liver involvement. These data suggest that in haemophilacs repeated and prolonged contact with the agent(s) responsible for post-transfusion hepatitis may cause chronic liver damage not associated with overt illness.

Effects of moderate consumption of red wine on platelet aggregation and haemostatic variables in healthy volunteers.

OBJECTIVE: To evaluate the effect of moderate consumption of red wine on platelet aggregation and haemostatic variables, discriminating the effect of alcohol from that of non-alcoholic components. DESIGN: A randomised crossover study. SETTING: The Department of Food Science and Technology, University of Milan. SUBJECTS: Eleven healthy male volunteers who were moderate drinkers. INTERVENTIONS: For three periods of four weeks, subjects drank three different beverages [320 ml of red wine (providing 30 g/day of alcohol), 30 g/day of alcohol diluted in 320 ml of clear fruit juice or 320 ml of dealcoholised red wine] during the two main meals. Each treatment was preceded by a period of four weeks of complete withdrawal from any alcoholic beverage. At the end of each period platelet aggregation after collagen and ADP stimulus, and levels of fibrinogen, plasminogen, tissue-type plasminogen activator (t-PA) antigen and von Willebrand factor (vWF) were determined. RESULTS: Consumption for a period of four weeks of 30 g/day of alcohol either from red wine or alcohol resulted in similar decreases of collagen-induced platelet aggregation and fibrinogen levels. ADP-induced platelet aggregation, t-PA antigen, vWF and plasminogen levels were not affected by any treatment. No differences were detected when we compared platelet function and the other haemostatic variables at the end of red wine and dealcoholised treatments with findings at the end of alcohol treatment and abstinence. CONCLUSIONS: The well known positive effect of moderate consumption of red wine on haemostasis seems due to alcohol and not to the non-alcoholic fraction present in red wine.

Composition of platelet phospholipids after moderate consumption of red wine in healthy volunteers.

OBJECTIVE: To evaluate the effect of moderate consumption of red wine on composition of platelet phospholipids, discriminating the effect of alcohol from that of non-alcoholic components. DESIGN: A randomised crossover study. SETTING: The Department of Food Science and Technology, University of Milan. SUBJECTS: Eleven healthy male volunteers who were moderate drinkers. INTERVENTIONS: For three periods of 4 weeks, subjects drank three different beverages [320 ml of red wine (providing 30 g/day of alcohol), 30 g/day of alcohol diluted in 320 ml of clear fruit juice or 320 ml of dealcoholised red wine] during the two main meals. Each treatment was preceded by a period of 4 weeks of complete withdrawal from any alcoholic beverage. At the end of each period the fatty acid composition of individual phospholipids was determined on isolated platelets. RESULTS: Consumption for a period of 4 weeks of non-alcoholic components either from 320 ml of red wine or from the same amount of dealcoholised red wine resulted in similar increases in polyunsaturated fatty acids in all phospholipid fractions of platelet, with the exception of sphingomyelin. No differences were detected when we compared the composition of phospholipids at the end of red wine and alcohol treatments with findings at the end of dealcoholised treatment and abstinence. CONCLUSIONS: The increase of polyunsaturated fatty acids in platelet phospholipids due to the non-alcoholic components of red wine suggests an antioxidant effect that could be relevant in justifying the protective effect of red wine shown in epidemiological studies.

Platelet coagulant activities and serum lipids in transient cerebral ischemia.

To determine whether platelets play a part in the pathogenesis of transient cerebrovascular ischemia, we studied 22 patients with transient ischemia, 18 control patients and 38 normal subjects. Platelet aggregation and [14C]-serotonin release by ADP, epinephrine and collagen were normal in all patients, as were plasma coagulation assays, except for shortened partial thromboplastin times in the patients with transient ischemia. Platelet coagulant activities concerned with initiation and early stages of intrinsic coagulation were increased two to three times in 12 patients with transient ischemic attacks with normal serum lipids and normal in the 10 others with Type IV hyperlipoproteinemia. These results indicate an association between platelet coagulant hyperactivity and transient ischemic attacks in a group of patients with normal serum lipids.

Acquired storage pool disease in platelets during disseminated intravascular coagulation.

A patient with clinical and laboratory evidence of disseminated intravascular coagulation associated with deep-vein thrombosis and pulmonary embolism developed a qualitative platelet abnormality characterized by a defective release reaction. Second-phase aggregation induced by ADP and adrenaline was impaired, and reduced collagen-induced aggregation was accompanied by defective release of ADP and ATP. The decrease in total platelet ATP and ADP, the high ATP:ADP ratio in the presence of normal amounts of metabolic adenine nucleotides, and the low content of serotonin associated with abnormal uptake and metabolism of the exogenous amine suggested that the defective platelet function was due to lack of the platelet organelles in which serotonin and nonmetabolic adenine nucleotides are normally stored. Acquired storage pool disease is likely to be related to exposure of circulating platelets to aggregating agents, with their degranulation occurring during disseminated intravascular coagulation.

Isolation and characterization of two domains of human von Willebrand factor that interact with fibrillar collagen types I and III.

We have identified four discrete proteolytic fragments of von Willebrand factor (vWF) that define two collagen-binding domains. Two of the fragments tested, T 96 kDa and T 55 kDa, were generated by digestion with trypsin, and two, Fragments I and III, with Staphylococcus aureus V8 protease. The larger Fragment III, a disulfide-linked homodimer, extends between residues 1 and 1365 of the 2050-residue vWF subunit and comprises the sequence of all the others. T 96 kDa, also a disulfide-linked homodimer, extends between residues 449 and 728. T 55 kDa and Fragment I, both single-chain polypeptides, have a partial sequence overlap corresponding to residues 911-1114, and together extend from residue 730 to 1365. The ability of the fragments to interfere with the vWF-collagen interaction was evaluated by measuring inhibition of 125I-labeled vWF binding to fibrillar bovine collagen types I and III. All the four fragments tested inhibited binding. Native conformation was essential for expression of this function; denaturation with guanidine hydrochloride and reduction of disulfide bonds resulted in marked reduction or complete loss, respectively, of the inhibitory activity at all the concentrations tested. Two monoclonal antibodies were prepared, one directed against T 96 kDa and the other against Fragment I. Both antibodies partially inhibited vWF binding to collagen, and their inhibitory effect was enhanced when they were used together. 125I-Labeled Fragment I bound to collagen in a saturable manner, and the binding was completely blocked by both T 96 kDa and T 55 kDa. Thus, we have identified at least two distinct functional domains of vWF that concurrently mediate the vWF-collagen interaction. The two domains appear to share a common recognition site on collagen.