A B ST R A CT The binding of '251-von Willebrand factor to platelets stimulated by thrombin, ADP, and a combination of ADP + epinephrine (EPI) is specific, saturable, and reversible. Active platelet metabolism and divalent cations are required for binding induced by these stimuli, but not by ristocetin, suggesting the existence of different mechanisms involved in the vWF-platelet interaction. A monoclonal antibody directed against an epitope of membrane glycoprotein (GP) lb had no effect on the binding of '251-vWF to normal platelets stimulated by thrombin or a combination of ADP + EPI, but completely blocked ristocetin-induced binding. Binding induced by thrombin to GPIb-blocked platelets was specific. Moreover, thrombin-induced binding of 1251-vWF was increased, rather than decreased, in two patients with the Bernard-Soulier syndrome whose platelets lacked GPIb. Conversely, monoclonal antibodies directed against the GPIIb/IIIa complex had no effect on ristocetininduced binding of (vWF)' is a large multimeric glycoprotein that circulates in blood complexed with the Factor VIII procoagulant activity protein (1). It plays an essential role in platelet function as shown by the prolonged bleeding time in von Willebrand disease (1). Specific binding sites for vWF are induced on the platelet membrane by the antibiotic ristocetin (2), a nonphysiologic agent, as well as by thrombin (3, 4) and ADP (5). Platelet membrane glycoprotein (GP) lb is considered to function as the surface receptor for vWF (6). In the Bernard-Soulier syndrome, a congenital bleeding disorder, platelets lack GPIb (7) and the ristocetin-induced binding of vWF is decreased (8). On the other hand, in Glanzmann thrombasthenia, another congenital platelet abnormality, there is a marked decrease of the membrane GP complex Ilb/Illa but normal content of GPIb (9). The ristocetin-induced binding of vWF to thrombasthenic platelets has been reported to be normal (8). In contrast, we have recently shown that binding of vWF to thrombasthenic platelets stimulated by thrombin is severely deficient (4). Therefore, we postulated that different sites are involved in ' Abbreviations used Marguerie et al. (12). The final platelet suspensions were all in modified calcium-free Tyrode buffer, containing 137 mM NaCI, 2 mM MgCI2, 0.42 mM NaH2PO4, 11.9 mM NaHCO3, 2.9 mM KCI, 5.5 mM glucose, 10 mM Hepes, pH 7.35, and 20 mg/ml bovine serum albumin (Fraction V, CalbiochemBehring Corp., American Hoechst Corp., San Diego, CA). When experiments of serotonin release were performed, PRP was labeled before washing by incubating 20 ml with 0. (3.3,M). However, ADG-washed platelets usually gave reversible aggregation to ADP and were unresponsive to epinephrine (EPI). On the contrary, gel-filtered platelets always gave irreversible aggregation to ADP, and responded to 20 MM EPI in the presence of fibrinogen (3.3 ,M) with a typical two-wave aggregation. Contamination of plasma vWF in all washed platelet preparations was below 5 X 10' U/dl (1 dl of a normal plasma pool contains ...

SummaryA polyelectrolyte-fractionated porcine factor VIII concentrate was given to 16 hemophiliacs with anti-F VIII antibodies (Ab) and to a woman with post-partum-acquired Ab during 24 courses of treatment including three major surgical procedures. Before treatment, antiporcine F VIII Ab was always lower than antihuman F VIII Ab, with a median cross reactivity of 32%. After treatment, the mean rise in F VIII was 1.5 U/dl/Unit infused/Kg b.w. and in vivo recovery was 50% of the theoretical values. Anamnestic rises in anti-porcine F VIII Ab (3 × the baseline titer) were seen after 9 of 22 courses of treatment with porcine F VIII only; similar rises in anti-human F VIII Ab, after 6 courses of treatment; median cross reactivity did not change significantly. Lower than expected increases in plasma F VIII without marked changes in Ab titers and severe thrombocytopenia occurred during surgery in two patients. Porcine F VIII is a rational and effective therapeutic choice for patients who have anti-human Ab titers above 10 U/ml; it can solve clinical situations that would otherwise be very difficult to manage; anamnesis is perhaps less frequent than after human F VIII; however, the incidence of thrombocytopenia, resistance and other side- effects is still higher than desirable.

Nineteen pregnant women with uncomplicated pregnancies were studied during the first, second, and third trimesters. We measured the following hemostatic parameters: prothrombin time, activated partial thromboplastin time, fibrinogen, antithrombin III, protein C, protein S, platelet number and volume. Platelet function was examined by a cytofluorimetric method, using an anti-GPM-140 antibody which is directed against a platelet alpha granule membrane protein. Activated platelets were expressed as a percentage of the GMP-140-positive platelets over total platelets. Fibrinogen levels showed a steady increase during pregnancy; conversely prothrombin time, activated partial thromboplastin time, protein C, and antithrombin III showed no significant modifications and remained within the reference range. There was a decrease of protein S activity throughout pregnancy, although protein S antigen did not follow this trend. The decrease occurred early in pregnancy and persisted during the second and third trimesters, reaching a stable plateau. We observed no platelet volume change or activation: the percentage of activated platelets was within the normal reference range, even in late pregnancy.

In spite of their recognized risk of thrombosis, patients with myeloproliferative neoplasms (MPN) show little or no abnormalities of traditional coagulation tests, perhaps because these are unable to represent the balance between pro- and anticoagulants nor the effect of platelets and blood cells. We investigated whether global tests such as thrombin generation in platelet-rich plasma (PRP) or thromboelastometry in whole blood were able to detect signs of procoagulant imbalance in MPN. The endogenous thrombin potential (ETP) of 111 patients and 89 controls was measured in PRP with platelet count adjusted to the original patient- or control-count. Testing was performed with and without thrombomodulin (the physiological protein C activator) and results were expressed as ETP ratios (with/without thrombomodulin). High ETP ratios reflect resistance to thrombomodulin and were taken as indexes of procoagulant imbalance. Patients were also investigated by thromboelastometry that provides such parameters as the clot formation time (CFT) and maximal clot firmness (MCF). Short CFT or high MCF were taken as indexes of procoagulant imbalance. ETP ratios were higher in patients than in controls and were directly correlated with platelet counts and inversely with the plasma levels of free protein S, protein C and antithrombin. Patients on hydroxyurea had lower ETP ratios than those on other treatments. CFT was shorter and MCF was greater in patients than controls; CFT and MCF were correlated with platelet counts. In conclusion, patients with MPN display a procoagulant imbalance detectable by thrombin generation and thromboelastometry. These tests might be useful in the frame of clinical trials to assess their association with the occurrence of thrombosis and with the effect of therapeutic strategies in MPN.

Background and Purpose-The etiology of germinal matrix hemorrhage-intraventricular hemorrhage (GMH-IVH) is multifactorial and the role of genetic polymorphisms is unclear. The aim of this prospective study was to evaluate prothrombotic genetic mutations as independent risk factors for the development of all grades of GMH-IVH in very-low-birth-weight infants. Methods-The presence of both factor V Leiden and prothrombin gain-of-function gene mutations were prospectively assessed in 106 very-low-birth-weight infants.

Platelet function was investigated in full-term infants on the first, the fourth and the tenth days of life and compared to normal adult controls. Platelet function was analyzed through a new cytofluorimetric technique with two murine monoclonal antibodies, PAC-1 and anti-GMP-140, directed against two membrane proteins expressed on the activated platelets’ surface. The percentage of activated platelets detected with PAC-1 and anti-GMP-140 was evaluated at basal condition and after in vitro stimulation with a weak agonist (ADP) and a strong Txa₂ analogue inducer (U 46619). At day 1 platelet activation at basal condition was negligible and similar to adult controls both with PAC-1 (1.2 vs. 1.1%) and anti-GMP-140 (2.6 vs. 3.3%). On the contrary, after ADP stimulation the percentage of PAC-1-positive activated platelets was significantly reduced in neonates compared to adults (22 vs. 66%; p < 0.001) and even more after U 46619 (11 vs. 72%; p < 0.001). The percentage of anti-GMP-140-positive activated platelets behaved similarly after adding both ADP (26 vs. 46%; p < 0.01) and U 46619 (37 vs. 67%; p < 0.001). The reduced platelet activation after ADP and U 46619 persisted at day 4 both with PAC-1 and with anti-GMP-140. On the contrary, at day 10 newborn platelets analyzed with anti-GMP-140 behaved similarly to the adult ones both at basal condition and after stimulation with ADP or U 46619 (6 vs. 3% at basal state, 42 vs. 46% after ADP addition, and 55 vs. 67% after U 46619). These data demonstrate that the reduced platelet activation present in newborns is restored by the tenth day after birth.

The abnormal multimeric composition of plasma von Willebrand factor in type IIB von Willebrand's disease is transiently corrected after infusion of 1-deamino-[8-D-arginine]-vasopressin. However, the larger multimers released into the circulation disappear more rapidly in these patients than in type I von Willebrand's disease or normals. We demonstrate that the larger multimers of normal von Willebrand factor transfused into a type IIB patient are cleared from the circulation more slowly than multimers of similar size endogenously released from tissue stores. The rate of disappearance of large von Willebrand factor multimers after infusion of cryoprecipitate is similar in IIB, IIA, and severe homozygous-like von Willebrand's disease. Platelets from the IIB patient exhibited normal ristocetin-induced binding of normal von Willebrand factor. However, like normal platelets, they bound IIB von Willebrand factor at lower ristocetin concentrations than required for normal von Willebrand factor. These findings provide evidence that absence of the larger multimers from IIB plasma is related to a molecular abnormality of von Willebrand factor rather than to enhanced affinity of abnormal tissue or cellular binding sites, as is the case in the recently described “pseud” von Willebrand's disease and “platelet-type” von Willebrand's disease.

Dear Sir, Long trips, especially air flights, are considered a risk for venous thromboembolism [1][2][3][4][5]. The frequency of symptomatic thromboembolic events increases with the distance flown, but appears to be low [3]. In a recent study, however, Scurr et al. found symptomless calf vein thrombosis detected by ultrasonography in up to 10% of long-haul travelers [4]. Given the limited sensitivity of the method [6], the true incidence of travel-associated distal thrombi may even be higher. Taken together, these data suggest that subclinical thrombosis may be frequent during air travel but usually resolves once mobility is resumed. Accordingly, symptomatic thromboembolic events would reflect a rare situation where activation of coagulation escapes the natural control systems.The prolonged sitting position during an air trip is associated with venous stasis, decreased blood flow and hemoconcentration, and is widely assumed to be the main causal factor for thrombus formation in this setting. If this assumption is correct, prolonged immobility in a sitting position by itself would be sufficient to activate coagulation. Subclinical activation of coagulation À a so-called prethrombotic state [7] À can be assessed by sensitive biochemical markers of thrombin generation (prothrombin fragment 1 þ 2, F1 þ 2), of ongoing fibrin formation (fibrinopeptide A, FPA) and lysis (D-dimer, DD). The aim of this study was to test the hypothesis that prolonged immobility in a sitting position similar to the one occurring during air traveling activates coagulation.Forty healthy subjects of either sex (median age 29.5 years, range 19-58) with a negative personal and family history and no known risk factor for thromboembolism were kept seated for 6 h on normal chairs, with their knees bent; during this period they were allowed to drink, eat, read or watch television. At baseline and after 3 and 6 h of immobilization, blood was collected in CTADPPACK anticoagulant from sitting probands with a clean venepuncture from an antecubital vein under controlled venous stasis for measurement of F1 þ 2 (Enzygnost F1 þ 2 Behring), FPA (RIA reagents supplied by Imco), and DD (mini-VIDAS, BioMérieux). Hemoglobin concentration and hematocrit were determined as well. In order to control for the circadian variations of the hemostatic system [8], we used the same protocol for blood sampling in a subgroup of 18 volunteers who were allowed to move freely (control group).Statistical probability was assessed with repeated measures ANOVA on ranks followed by pairwise multiple comparison procedures (Student-Newman-Keuls method) for non-normally distributed data. A linear regression was done for correlation between data at baseline and after 6 h. A difference was considered significant when P <0.05.Over the 6 h of sitting position there was no change in the plasma levels of FPA and DD, whereas F1 þ 2 levels decreased from a median of 0.49 to 0.40 nmol L À1 (P < 0.0001, Table 1).The F1 þ 2 decrease was both relevant in quantitative terms (À20% of the baseline ...