[A modified in vivo platelet retention test in the evaluation of primary hemostasis]. / Ein modifizierter In-vivo-Plättchenretentionstest zur Beurteilung der primären Hämostase.

A simple assay system for measuring platelet retention in a standardized superficial skin wound is presented. Platelets were counted in the blood collected at the site of the wound 1, 2 and 3 minutes following incision. Per cent platelet retention was calculated from the difference between venous blood platelet count and wound blood platelet count, divided by venous count. The time course of platelet retention, measured in 20 healthy persons, reflected the sequence of primary and secondary platelet aggregation. Ten patients with congenital platelet defects already showed impaired platelet retention in the early stage of bleeding. On the other hand, administration of aspirin to 10 healthy subjects inhibited only the late stage of platelet retention. In comparison with the template bleeding time our platelet retention test provides additional information useful for evaluating congenital and drug-induced defects in platelet plug formation.

Monitoring of heparin therapy with the activated partial thromboplastin time and chromogenic substrate assays.

Heparin therapy was monitored with the activated partial thromboplastin time (APTT) and with chromogenic substrate assays (factor Xa and factor IIa inhibition) in 100 plasma samples from 47 patients. Heparin concentrations were classified as being below, within or above a defined therapeutic range (TR; 0.2-0.55 units heparin/ml). In a first group of patients (A), all three assays allocated the plasma heparin levels to the same concentration interval with respect to the TR. The most frequent diagnoses in group A were uncomplicated arterial or venous thromboembolism, myocardial infarction with limited tissue necrosis, cardiac surgery without major complications and successfully treated infectious disease. In a second group of patients (B), the results of APTT suggested higher heparin concentrations with respect to the TR than the chromogenic assays. Predominant diagnoses were severe infectious diseases, severe liver disorders, extensive myocardial infarction and postoperative complications after cardiac surgery. The discrepancy between heparin concentrations determined by either APTT or the chromogenic substrate assays is most likely due to a non-heparin related prolongation of APTT caused by the underlying disease.

[Blood coagulation disorders]. / Störungen der Blutstillung.

Disorders of hemostasis are frequently encountered, and, if not recognised as early as possible, in particular prior to surgery, may lead to bleeding complications which are much more difficult to treat than those where exact diagnosis permits specific preventive therapy. A first case report on a woman with an acquired inhibitor against factor VIII illustrates that even minor injury may lead to hemorrhage necessitating blood transfusion. Furthermore, as long as the defect remains inaccessible to successful substitution therapy, wound healing will not take place. A second case report describes a patient with a congenital dysfunction of blood platelets, and serves as an example for similar disorders which, if unrecognised, lead to multiple, otherwise avoidable complications. Unfortunately there is no clearcut correlation between a given defect of hemostasis and bleeding manifestations, since psychological factors, among others, appear to influence the occurrence of "spontaneous" hemorrhage. Any unexpected and unexplained bleeding complication requires careful evaluation of the possible underlying causes, since such investigations often not only affect the future of the propositus himself but also that of his family members.

Exposure of platelet binding sites in von Willebrand factor by adsorption onto polystyrene latex particles.

Von Willebrand factor molecules are flexible linear polymers composed of repeating protomeric polypeptide subunits. In the process of primary hemostasis, von Willebrand factor promotes platelet adhesion and platelet plug formation at the site of vascular injury. This biologic activity is apparently related to the multimeric size of von Willebrand factor. We simulated von Willebrand factor binding to the subendothelial surface by adsorbing purified human von Willebrand factor onto polystyrene latex particles of two different diameters, i.e., 0.312 micron and 2.02 micron. The rate and extent of 125I-labeled von Willebrand factor binding to polystyrene was similar with both size classes of latex particles. The von Willebrand factor-coated latex beads of 2.02 micron diameter, in contrast to the smaller size, induced rapid agglutination of formalin-fixed human platelets in the absence of any other aggregating agent. Von Willebrand factor was also adsorbed from human plasma onto latex particles coated with anti-von Willebrand factor antibodies. Again, only the large beads, carrying the von Willebrand factor-antibody complex, induced agglutination of fixed platelets. Shear stress promoted the rate of von Willebrand factor adsorption to latex particles. Our results suggest that adsorption to surface exposes binding sites in human von Willebrand factor for platelets.

Three abnormal fibrinogen variants with the same amino acid substitution (gamma 275 Arg----His): fibrinogens Bergamo II, Essen and Perugia.

We report on three unrelated individuals with the same uncommon type of dysfibrinogenemia, originating from Bergamo, Essen and Perugia. None of them showed bleeding symptoms while the Bergamo patient and members of her family presented with a thrombotic tendency. The presence of a defective fibrinogen was suggested by prolonged thrombin and reptilase times. Furthermore, fibrinogen concentrations of less than 0.28 g/L were determined by the functional assay whereas values of 1.5-2.4 g/L were measured by heat precipitation or electroimmunoassay. Fibrinogen was isolated by affinity chromatography on insoluble fibrin monomer. The rate of fibrinopeptide release by thrombin was normal while the fibrin polymerization reaction was strongly delayed. An abnormal peptide (gamma 265-310) was isolated by high-performance liquid chromatography after cyanogen bromide cleavage of the purified gamma-chain of fibrinogen Bergamo II and Essen. The same peptide was also isolated following cyanogen bromide treatment of the intact fibrinogen Perugia. Sequence analyses of these peptides demonstrated the same amino acid exchange in all three fibrinogens: gamma 275 arginine----histidine. The described fibrinogen variants appear to possess a molecular defect which has thus far only been observed in fibrinogen Haifa.

[Monitoring heparin therapy by thrombin time and activated partial thromboplastin time--a comparison]. / Uberwachung der Heparintherapie mit der Thrombinzeit und der aktivierten partiellen Thromboplastinzeit--ein Vergleich.

In 106 plasma samples obtained from patients on heparin therapy, monitoring by 2 methods (activated partial thromboplastin time and thrombin clotting time--APTT and TT) was compared. All patients in whom APTT indicated markedly higher plasma heparin concentrations than the TT were critically ill (group B): their main diagnoses included severe infectious disease, severe liver disease and extensive myocardial infarction. Patients with lesser discrepancies between the results of APTT and TT did not suffer from such severe conditions (group A). Cardiac surgery without major postoperative problems, limited myocardial infarction and uncomplicated thromboembolism were the main diagnoses in this group. In group B, non-heparin related prolongation of APTT was thought to be the main factor responsible for the overestimation of plasma heparin concentrations by this test. We conclude that in patients with severe infectious disease, liver disease or extensive tissue necroses (i.e. myocardial infarction), APTT cannot be recommended for laboratory monitoring of heparin therapy.

[A new test for evaluating the von Willebrand factor: collagen-induced agglutination of fixed thrombocytes]. / Ein neuer Test für die Beurteilung des von Willebrand-Faktors: kollagen-induzierte Agglutination fixierter Thrombozyten.

In primary hemostasis von Willebrand factor (vWf) promotes the adhesion of platelets to subendothelial structures of the damaged blood vessel. A new test is described which measures the collagen binding affinity of vWf. Activities of vWf in plasmas of 51 patients were measured with either collagen (ColCof) or ristocetin (RCof). Regression-analysis showed a good correlation of both activities (r = 0.924). In a second group of 21 patients with a variant of von Willebrand's disease, ColCof activity was generally higher than RCof activity.

[Binding of the von Willebrand factor to solid surfaces: effect of shear strength]. / Bindung des von Willebrand-Faktors an feste Oberflächen: Einfluss der Scherkraft.

Platelet adhesion onto subendothelium of a damaged blood vessel depends upon the presence of von Willebrand factor (vWf) only at high flow shear rate. Our results showed enhanced adsorption of vWf onto stainless steel and latex surfaces under conditions of high shear stress, thus suggesting that in the rapidly flowing blood the extremely long vWf molecules are stretched, thus accelerating their binding to a given surface.

Psychogenic purpura, idiopathic thrombocytopenic purpura, and platelet dysfunction in the same patient.

A patient whose peculiar and painful purpura seemed to be strongly related to psychogenic factors is described. The skin bleeding pattern in this patient was consistent with the diagnosis of psychogenic purpura (autoerythrocyte sensitization). In addition, idiopathic thrombocytopenic purpura and platelet storage pool deficiency were present. These somatic conditions are considered to be of minor importance in the pathogenesis of this hemorrhagic syndrome. The association of these various types of bleeding disorders in the same patient has not been previously described.

Characterization of fibrinogen Milano I: amino acid exchange gamma 330 Asp----Val impairs fibrin polymerization.

An abnormal fibrinogen was found in two asymptomatic members (father and daughter) of the same family, originating from northern Italy. Routine coagulation studies revealed prolonged thrombin and reptilase clotting times. Plasma fibrinogen levels, as determined by a functional assay, were markedly diminished, whereas the heat precipitation method indicated normal fibrinogen values. On the basis of these findings, a tentative diagnosis of dysfibrinogenemia was made, and according to the accepted nomenclature, this fibrinogen variant was called "fibrinogen Milano l." The time course of fibrinopeptide A and B release from fibrinogen Milano l was normal, but the aggregation of fibrin monomers was delayed. Two-dimensional electrophoresis of reduced variant fibrinogen chains showed a defective gamma-chain with increased cathodic mobility. An abnormal electrophoretic mobility was observed also for the gamma-chain remnants of fibrinogen fragments D1 and D2 derived from fibrinogen Milano l, whereas the charge anomaly was lost after a further digestion by plasmin to D3, suggesting that the structure abnormality of this variant is situated in the region gamma 303-356. An abnormal peptide was isolated after cyanogen bromide cleavage of intact fibrinogen Milano l. This fragment spans from position gamma 311 to gamma 336. Amino acid analysis of the abnormal peptide showed the presence of valine and a diminished content of aspartic acid. Sequence analysis demonstrated an amino acid exchange Asp----Val in the gamma-chain at position 330.

Von Willebrand factor-dependent agglutination of washed fixed human platelets by insoluble collagen isolated from bovine aorta.

Adult bovine aortic tissue was homogenized in a neutral phosphate buffer containing proteinase inhibitors. The insoluble residue was rehomogenized in Tris-buffered 6 mol/L guanidinium chloride (pH 7.4). An insoluble fibrillar protein, floating above the main pellet after recentrifugation, was harvested. This material agglutinated washed fixed human platelets in the presence of either normal human plasma or purified von Willebrand factor (vWF). No such reaction was seen when either buffer or plasma from patients with severe von Willebrand's disease was added instead. The extent of platelet agglutination was measured photometrically, similarly to the ristocetin cofactor assay. The agglutination reaction was strongest at neutral pH and was impaired after either addition of EDTA or previous digestion of the fibrillar material by collagenase or pepsin. By light microscopy platelets were seen to adhere onto isolated fibers. Amino acid composition, subunit polypeptides, substrate properties, and interaction with fibronectin of this fibrillar protein were comparable to those of collagen. Therefore, we tentatively denote the induction of platelet agglutination by vWF protein in the described test system as "vWF-collagen cofactor" activity. Comparison of this activity in 65 plasma samples, containing various concentrations of vWF, with ristocetin cofactor activity showed good correlation between results obtained in both tests (r = 0.91).

[How can we improve long-term anticoagulation?]. / Wie können wir die Langzeit-Antikoagulation verbessern?

Oral anticoagulation is sometimes unjustly referred to as a particularly difficult form of antithrombotic therapy. Apparent failures of this treatment may be caused by insufficient information on the part of either the physician or the patient himself, poor standardization of laboratory tests and/or inadequate dosage of vitamin K antagonists. Specialized centers for treatment of thrombosis have done pioneer work in standardizing and evaluating oral anticoagulant treatment with respect to various indications. Based on this experience, optimum long-term anticoagulant therapy is today possible even in a small hospital or in general medical practice, provided that the pharmacological peculiarities of vitamin K antagonists and international developments concerning standardization of the prothrombin time (Quick test) and its modifications (International Normalized Ratio, INR) are taken into consideration. Regular internal and external quality control of laboratory tests for monitoring of oral anticoagulation is of the utmost importance.

Reactivity of small molecular forms of human factor VIII/von Willebrand factor with botrocetin and anti-factor VIII-coated latex particles.

Two recently developed tests for measurement of factor VIII/von Willebrand factor (FVIII/vWF), i.e. platelet agglutination by botrocetin and a kinetic latex antigen assay, were compared with ristocetin cofactor and electroimmunoassay, in respect with FVIII/vWF size-distribution. FVIII/vWF was measured in six cases of atypical von Willebrand's disease (type II), in gel-filtered fractions of normal cryoprecipitate and in the course of depolymerization of purified normal FVIII/vWF by disulfide reduction. Small molecular forms of FVIII/vWF from normal and variant type II plasma, and those derived by disulfide reduction of high-molecular weight FVIII/vWF, showed remarkably decreased reactivity in ristocetin-, botrocetin- and latex-assay. We conclude that for botrocetin-induced platelet agglutination, as well as for agglutination of antibody-coated latex particles, multiple interactions with repeating subunits of FVIII/vWF are required. As a practical consequence, the combined measurement of FVIII/vWF by the latex test and electroimmunoassay provides a simple tool for discriminating between the classical von Willebrand's disease and its variants.