E. Posan

Comparison of PFA-100 testing and bleeding time for detecting platelet hypofunction and von Willebrand disease in clinical practice

The PFA-100 instrument (Platelet Function Analyzer, Dade Behring) has been reported to be superior to the bleeding time (BT) as a screening test of primary hemostasis. However evaluation of this device has been principally limited to selected populations. The studys aim was to determine testing performance in clinical practice, by comparing the PFA-100 to the BT for the identification of von Willebrand disease (VWD) and intrinsic platelet hypofunction. From 1998-2000, PFA-100 closure time (CT) for epinephrinecollagen (EPI) and ADP-collagen (ADP) cartridges and modified Ivy BTs were performed on outpatients referred for testing for suspected or known hemorrhagic diathesis (n = 346). Evaluation included assays of von Willebrand factor and platelet aggregometry in addition to platelet flow cytometry and electron microscopy when indicated. The normal distribution of PFA-100 CTs was determined using blood samples from 61 normal donors studied on 155 occasions. Results show that thirty-four patients met the diagnostic criteria for VWD and 31 patients were diagnosed with congenital or acquired intrinsic platelet hypofunction. The sensitivity of the PFA-100 for identification of VWD was significantly better (p < 0.01) than the BT with similar specificity. In contrast, the PFA-100 was comparable, but not superior to the BT for detecting platelet hypofunction. We conclude that the PFA-100 performance compares favorably to the BT for the identification of intrinsic platelet hypofunction in clinical practice with superior sensitivity for detecting VWD. Therefore, the PFA-100 could replace the BT for purposes of screening for VWD and intrinsic platelet hypofunction. When clinical suspicion is strong, testing should be supplemented with assays of von Willebrand factor and platelet aggregometry.

Thrombotic and fibrinolytic alterations in the aseptic necrosis of femoral head

&NA; Recent reports seem to support the role of the thrombophilia and decreased fibrinolysis in the aetiopathogenesis of aseptic necrosis of bone. In the present study, haemostatic disturbances were analysed in adults (n = 49) and patients in childhood (Perthes disease) (n = 47) with aseptic necrosis of the femoral head. Fibrinolytic parameters (in vitro clot lysis, plasminogen, plasmatic plasminogen activator inhibitor‐1 activity, D‐dimer) along with lipoprotein (a) [Lp(a)] and fibrinogen were measured. von Willebrand factor, platelet activation and some thrombophilic factors (activated protein C resistance and factor V Leiden mutation, protein C, protein S activity) were also determined. Impaired fibrinolysis, an increased Lp(a) level along with slow clot lysis and increased platelet activation were found in adult cases. We detected five cases of factor V Leiden mutations (one heterozygotic and four homozygotic) among patients with Perthes disease. The clinical course of the heterozygous case was similar to the usual form of Perthes disease. The most severe form of Perthes disease has been observed in homozygous factor V Leiden mutation cases. The mutation of factor V Leiden per se probably does not induce the development of aseptic necrosis of bone tissue in childhood, but it does play a role in its acceleration. Homozygous factor V Leiden mutation definitely runs a more severe course. On the other hand, in adult cases, the disturbances of haemostasis, impaired fibrinolysis, elevated Lp(a) level, increased platelet activation and slight elevation of fibrinogen might have clinical relevance. Further studies should focus on proving the role of the haemostatic alterations in the pathogenesis of severe forms of aseptic bone necrosis. The use of antithrombotic drugs in order to slow the process of aseptic necrosis also has to be addressed in future surveys. Blood Coagul Fibrinolysis 14:243‐248

Coumarin-Induced Skin Necrosis Following Heparin-Induced Thrombocytopenia and Thrombosis A Case Report

A severe thrombotic thrombocytopenic status, induced by heparin in a sixty-nine-year-old woman undergoing total hip joint arthroplasty, was treated by switching the anticoagu lant therapy to coumarin, which induced skin necrosis. There appeared to be a possible causal relation between the severe immune reaction to heparin and the condition that predisposed to skin necrosis in the presence of coumarin. In patients who express a strong immune response to heparin, a different anticoagulant approach other than use of coumarin congeners appears to be justified..

Effect of L-arginine on in vitro plasmin-generation and fibrinogenolysis*

L-arginine ahs received much attention in numerous aspects of the regulation of vascular tone and haemostasis. L-arginine seems to be capable to bind to plasminogen, too. The aim of the present paper is to investigate the action of L-arginine on in vitro plasmin generation and fibrino(geno)lysis by chromogenic, kinetic plasmin generation assay and electrophoretic analysis. The acceleration of tPA-induced plasmin generation in the presence of low concentration of L-arginine, along with augmentation of in vitro fibrinogenolysis have been documented. L-arginine may have a role in the modification of fibrinogenolysis, and this role should be considered if arginine is used as an element of some novel antithrombotic agents.

Increased platelet activation and decreased fibrinolysis in the pathogenesis of aseptic necrosis of the femoral head.

Thrombotic events, increased tendency toward intravascular thrombosis and decreased fibrinolysis seem to be possible pathologic causes for aseptic necrosis of the femoral head (ANFH) in adults. This project was to study whether either increased platelet activation or decreased fibrinolytic activity and/or any other thrombogenic factor may be implicated in the evolvement of ANFH. The speed of the in vitro lysis was significantly lower in patients (both in primary and in secondary cases) compared with healthy controls. The platelet activation (measuring with beta TG) proved to be significantly higher in the primary group as well as in the secondary group compared with healthy controls. Lp(a) levels were elevated in primary and secondary cases. This alteration was more characteristic in the primary cases. Fibrinogen levels were also elevated in the primary group, but the difference was not significant. The data shown here may further support that hypofibrinolysis and increased thrombogenesis are major causes of ANFH. Early diagnosis of ANFH increases the chances of modifying the course of this disabling disease.

Altered lysis resistance of platelet-rich clots in patients with insulin-dependent diabetes mellitus

The fibrinolytic resistance of platelet-rich arterial thrombi received much attention. Clot lysis method was used to assess the in vitro fibrinolytic properties in diabetes mellitus. Platelet rich (PRP) clots were formed by addition of thrombin, and lysis was induced by tissue-plasminogen-activator. The coagulation and lysis was followed by the light scattering properties. A special pattern of good initial lysis followed by a second clotting phase was observed in more than half of insulin dependent diabetic patients, while a similar pattern of clot-lysis was only occasionally found in non-insulin dependent diabetes mellitus or in the healthy control group. Following the thrombin activation of washed, gel-filtered platelets, the supernatants possessed an inhibitory action on in vitro lysis of PPP-clots. This suppression was remarkably stronger in IDDM, along with the highest PAI-1 activity concentration ratio of the platelet lysates, compared to plasmatic levels. The relation of this special type of PRP clot-lysis resistance to diabetic vascular complications needs further clarifying and investigations.

RGDFAP: platelet aggregation inhibitory and profibrinolytic hybrid peptide (RGDF coupled with the carboxy terminal part of alpha 2-antiplasmin) enhances plasminogen binding to platelets.

As published in a recent issue of Blood Coagulation and Fibrinolysis, the hybrid peptide RGDFAP, composed of RGDF (Arg-Gly-Asp-Phe) coupled to a synthetic peptide residue of the carboxy terminal part of antiplasmin (AP26) inhibited platelet activation and augmented plasmin generation and in vitro fibrin clot lysis. This peptide contains an RGD motif which provides linkage to platelet GP IIb-IIIa. The antiplasmin part of the molecule may attach free plasminogen, which in turn increases the amount of platelet surface bound plasminogen, probably yielding enhanced lytic action at the site of thrombus formation. This hypothesis was investigated and confirmed by the results of platelet-plasminogen binding assays, using FITC-labelled antiplasmin antibodies and radioligand binding analysis. Increased platelet-linked plasminogen was detected by a chromogenic method, along with the acceleration of in vitro lysis of platelet-rich clots in the presence of RGDFAP peptide.

Possible role for platelet insulin receptors in modulating platelet function in health and diabetes mellitus

Binding studies have shown that human platelets contain binding sites for insulin with a surface density similar to that described for other cells.(1) Evidence for a reduced number and affinity of human platelet membrane insulin binding sites in non-insulin dependent diabetes mellitus (NIDDM) has been provided.(2) However, the influence of insulin and insulin receptors on platelet function has not been completely investigated and clarified. Falcon et al(3) reported phosphorylation of a subunit of the insulin receptor on platelets in response to insulin, but no alterations were detected in cAMP formation or degradation, inositol phosphate formation or phosphorylation of proteins other than the receptor itself. On the other hand Trovati et al(4) found reduced platelet aggregation responses to ADP, PAF, epinephrine, collagen and arachidonate in the presence of 40 µU/ml insulin. Their experience with an euglycemic-hyperinsulinemic clamp provided some further in vivo evidence to support the above mentioned findings. Platelet thromboxane A(2) formation was not altered in the presence of the hormone.

New pentamidine related substances which simultaneously inhibit platelet aggregation and accelerate plasmin generation and in vitro clot lysis

Pentamidine, a highly toxic drug, possesses RGD-peptide (Arg-Gly-Asp)-like antiplatelet actions. The objective of this investigation was to study the anticipated profibrinolytic and antiplatelet actions of pentamidine and of pentamidine (bearing guanidino-like groups)-related synthetic peptidomimetic compounds. Platelet aggregation inhibition was assessed using ADP, thrombin, collagen, arachidonic acid and epinephrine as inducers, by aggregometry. In vitro chromogenic plasmin generation tests and clot lysis assays were also performed. Two (assigned as D-2 and D-3) of the synthetic pentamidine-guanidino related molecules were able to inhibit platelet aggregation and simultaneously accelerate in vitro plasmin generation and clot-lysis in the nM range. These dual action antithrombotic agents now need to be tested further to assess their antithrombotic actions in vivo.