[Haemostatic testing prior to elective surgery? Yes!]. / Laboranalytischer Ausschluss einer hämorrhagischen Diathese vor elektiven Eingriffen? Ja!

Haemorrhagic disorders must be excluded prior to any operation or other invasive procedure that has the potential to involve serious bleeding. When assessing the individual risk of bleeding, screening tests of hemostasis must be combined with the patient's clinical history and symptoms, and any history of bleeding must be explored under direct medical supervision using a standardized questionnaire. However, this bleeding history is neither very specific, nor is it particularly sensitive. Screening tests that have been found to be useful include platelet count, activated partial thrombo plastin time (aPTT), prothrombin time (PT) and clottable fibrinogen. No reliable, sensitive and specific screening test is however available today to screen for platelet dysfunction or von Willebrand disease. A specialized coagulation laboratory should be involved when the bleeding history or laboratory screening indicate a potential haemorrhagic disorder.

Effects of desmopressin on platelet membrane glycoproteins and platelet aggregation in volunteers on clopidogrel.

PURPOSE: The use of clopidogrel is standard in interventional cardiology. Haemorrhage occurs in some patients, which implies a need for a non-transfusional therapy. Desmopressin showed its efficacy as an antidote of acetylsalicylic acid. In this trial the effects of desmopressin on platelet glycoproteins and the platelet's ability to aggregate under the influence of clopidogrel are studied. METHODS: The trial was conducted as an open, prospective, single-centre, randomised pilot study with n=17 healthy volunteers in a parallel-group design. 1 h after an oral loading dose of 375 mg clopidogrel the effects of a single-dose of 300 mug of Octostim nasal spray (n=9) on platelet aggregation, activity of platelets on the density of membrane-bound receptors are measured. RESULTS: Ristocetin cofactor and platelet reactivity rose significantly after the administration of Octostim nasal spray with 31.9% and 5.3%, respectively (p=0.0329; p=0.0414). The ADP-induced platelet aggregation increased after the administration of Octostim nasal spray by approximately 20% (p=0.0564). The fraction of CD62- and CD63-positive platelets did not change after clopidogrel nor after desmopressin (p=0.4203; p=0.6774). The density of GPIIb/IIIa receptors per platelet did not change after desmopressin (p=0.9652). The density of GPIb/IX receptors per platelet rose after desmopressin without reaching the level of significance (p=0.0802). In the desmopressin group alone the receptor density rose by 5.5% (p=0.0783). CONCLUSION: The administration of desmopressin improved the primary haemostasis when given in addition to a clopidogrel therapy. Patients undergoing a heart catheter procedure with clopidogrel might benefit from the use of desmopressin when having a bleeding episode.

Preservation of in vitro function of platelets stored in the presence of a synthetic dual inhibitor of factor Xa and thrombin.

BACKGROUND: The use of citrate anticoagulant limits the clinical significance of platelet function tests. Thrombin inhibitors cannot prevent thrombin-induced platelet activation completely. We examined the influence of benzylsulfonyl-d-Arg-Pro-4-amidinobenzylamide (BAPA), a dual inhibitor of Factor Xa (FXa) and thrombin, on platelet responsiveness to agonists when measured between 2 and 24 h after venipuncture. METHODS: Blood samples from 36 individuals were anticoagulated with citrate and BAPA, respectively. Turbidimetric platelet aggregometry (TPA) and impedance platelet aggregometry (IPA), a whole blood platelet counting assay for measuring platelet aggregation (PCA), and Platelet Function Anlayzer-100 (PFA-100 closure times (CTs) were determined after whole blood storage between 2 and 24 h after venipuncture. Native whole blood was studied over 48 h to determine the inhibition of thrombin generation by BAPA, hirudin and melagatran. RESULTS: BAPA inhibited thrombin generation completely for 48 h, while hirudin and melagatran did not. The use of citrate resulted in significantly reduced TPA induced by arachidonic acid (AA) or adenosine 5'-diphosphate (ADP), and significantly reduced IPA regardless of agonist when measured 10 and 24 h after blood collection. PCA ratios in citrated blood also dropped significantly 10 and 24 h after venipuncture. The length of storage of BAPA-anticoagulated blood samples over 24 h had no significant influence on any platelet response. The reproducibility of platelet function assay results obtained from BAPA-anticoagulated samples was significantly better than corresponding data from citrated blood. CONCLUSION: TPA, IPA, PCA or PFA-100 CTs remain stable for 24 h when whole blood is anticoagulated with a dual inhibitor of FXa and thrombin. This would greatly simplify the shipment of samples for platelet function testing.

The impact of a medium molecular weight, low molar substitution hydroxyethyl starch dissolved in a physiologically balanced electrolyte solution on blood coagulation and platelet function in vitro.

BACKGROUND AND OBJECTIVES: Hydroxyethyl starches (HES) may have the potential to impact negatively on haemostasis. Recent findings suggest that side-effects on haemostasis stem not only from the physicochemical differences between HES, but also from the composition of the solvent. We compared the effects of a newly developed medium molecular weight (MW) and low molar substitution (MS) HES dissolved in a physiologically balanced electrolyte solution (MW 130, MS 0.42; B-HES) with a commercially available non-balanced HES (MW 130, MS 0.4; NB-HES), and with Ringer's lactate (RL) solution in vitro. MATERIALS AND METHODS: Activated partial thromboplastin time (APTT), factor VIII clotting activity (F VIII:C) and von Willebrand factor (vWF) activity were investigated in 48 healthy individuals. Platelet function as measured by turbidimetric platelet aggregometry and whole blood impedance aggregometry induced by adenosine diphosphate (ADP), collagen and thrombin receptor activating peptide (TRAP), and by ADP and TRAP-induced expression of activated platelet fibrinogen receptor glycoprotein (GP) IIb/IIIa was determined in 24 participants. Haemodilution (25% and 50%, v/v for blood coagulation analyses and 20% and 40%, v/v for platelet function studies) was performed using the two HES preparations and RL. RESULTS: APTT was significantly longer and F VIII and vWF significantly lower at 25% and 50% dilutions with NB-HES compared to B-HES and RL. At 20% and 40% dilutions, ADP and TRAP-induced expression of activated platelet surface GP IIb/IIIa was significantly increased by B-HES compared to NB-HES and RL. Percentages of platelet GP IIb/IIIa expression were also significantly greater in samples diluted with B-HES than in undiluted blood. Neither the diluent (B-HES, NB-HES and RL) nor the degree of dilution (undiluted, 20% and 40% dilution) had any significant influence on ADP, collagen or TRAP-induced turbidimetric platelet aggregation or impedance platelet aggregation. CONCLUSIONS: In contrast to a non-balanced 130 kDa, MS 0.4 HES (NB-HES), a 130 kDa, MS 0.42 HES preparation dissolved in a physiologically balanced electrolyte solution (B-HES) does not affect APTT, F VIII:C and vWF in vitro. Both types of HES do not affect platelet aggregation induced by ADP, collagen or TRAP. B-HES but not NB-HES increases the expression of activated platelet GP IIb/IIIa induced by ADP or TRAP.

Haemocompatibility of polymer-coated stainless steel stents as compared to uncoated stents.

Acute and subacute stent thrombosis still represent an unsolved problem in connection with endovascular stents. For this reason coatings are tested now with the intention to reduce thrombogenicity of stainless steel surfaces. This comparative study examined whether a polymeric stent coating affected the haemocompatibility of a stainless steel stent. For compatibility testing, coated and non-coated stents were implanted in a low-grade thrombogenic closed-loop system perfused with platelet rich plasma at shear rates far below the threshold value at which shear-rate-induced activation of thrombocytes occurs. After 21 circulations of the filling volume (exposure time: 6.2 min), the number of single circulating platelets in the perfusion system with uncoated stainless steel stents decreased almost twice as much as was the case with polymer-coated stents. This is thought to indicate that more thrombocytes had adhered to the uncoated stainless steel stent, or that the thrombocytes were clustered in circulating aggregates. Parallel to the platelet aggregation/adherence, a release reaction took place, as was evident from the TAT complexes indicating the generation of thrombin. In the case of the implantation of uncoated stainless steel stents, both the number of activated circulating thrombocytes and the level of platelet reactivity (number of thrombocytes circulating in the plasma as aggregates) were notably higher than in the system with polymer-coated stents. At the same time it should be noted that the activation or aggregation is almost wholly attributable to the exogenic surface of the implanted stent, since activation due to the tube system or to shear rate can be excluded (as shown by measurements of the system without a stent). In addition to activation of the thrombocytes, a notable increase in the number of receptors per platelet (significant only in the system with the uncoated stent) took place. This supports both the adherence of the thrombocytes and their readiness to aggregate, since more receptors (docking places for ligands) are available. The better haemocompatibility of the polymer-coated stents, as verified in the laboratory, was also evident under microscopic examination of the explanted stents following the perfusion tests.

Influence of the direct NO-donor SIN-1 on the interaction between platelets and stainless steel stents under dynamic conditions.

It was investigated whether the NO-donor SIN-1, the active metabolite of molsidomine, influenced the activation of platelets, the formation of circulating platelet aggregates, the spontaneous aggregation of platelets and the activation of the clotting system triggered by a body foreign surface in an in vitro closed-loop perfusion model. With human platelet-rich plasma at micromolar concentrations SIN-1 exerted pronounced effects on the interaction between platelets and an exogenous surface. In the absence of SIN-1, the number of circulating single platelets decreased significantly, which could be due either to the formation of circulating platelet aggregates or to the adhesion of platelets to the stent. Both these processes were blocked by the addition of SIN-1. Moreover, the platelets exhibited hyperaggregability in the absence of SIN-1 whereas the NO-donor was able to completely inhibit spontaneous platelet aggregation. Similar results were obtained in flow cytometry experiments. Without SIN-1, high platelet surface densities of both the GPIb/IX and GPIIb/IIIa receptors were observed. In addition, the density of the fibrinogen receptor increased significantly with the number of perfusion cycles. SIN-1 was able to suppress the augmented GPIIb/IIIa receptor expression significantly. Molsidomine seemed to have the potential to reduce the incidence of thrombotic processes triggered by the exogenous surface of the stent.

[Haemostatic disorders in ENT patients. Part 2: Pathophysiology, diagnostics, clinical feature and therapy]. / Störungen der Hämostase bei Patienten in der HNO-Heilkunde. Teil 2: Pathophysiologie,Klinik und Therapie ausgewählter Krankheitsbilder.

In the first part of the medical education article on haemostatic disorders in ENT patients the basic physiology of haemostasis and main diagnostic tools were presented and discussed. The second part presents disorders of the coagulation system,thrombocyte function and blood vessels with special emphasis on clinical practice in ENT surgery. In this context, haemophilia A and B, von Willebrand disease and different forms of thrombocytopenia are of main clinical importance. Some underlying diseases such as malignomas, renal and hepatic insufficiency in combination with drug therapy (e.g.anticoagulants and thrombocyte function inhibitors) play an important role in clinical practice as well. Sepsis and haemorrhage may lead to disseminated intravascular coagulation (DIC). Beside a systematic review, important haemostatic disorders are illustrated with case reports.

[Haemostatic disorders in ENT patients.Part 2: Pathophysiology, diagnostics, clinical feature and therapy]. / Störungen der Hämostase bei Patienten in der HNO-Heilkunde : Teil 2: Pathophysiologie,Klinik und Therapie ausgewählter Krankheitsbilder.

In the first part of the medical education article on haemostatic disorders in ENT patients the basic physiology of haemostasis and main diagnostic tools were presented and discussed.The second part presents disorders of the coagulation system,thrombocyte function and blood vessels with special emphasis on clinical practice in ENT surgery. In this context, haemophilia A and B, von Willebrand disease and different forms of thrombocytopenia are of main clinical importance. Some underlying diseases such as malignomas, renal and hepatic insufficiency in combination with drug therapy (e.g.anticoagulants and thrombocyte function inhibitors) play an important role in clinical practice as well.Sepsis and haemorrhage may lead to disseminated intravascular coagulation (DIC).Beside a systematic review, important haemostatic disorders are illustrated with case reports.

[Perioperative therapy of von Willebrand disease. Demonstration of pathophysiology, clinical problems and therapy options using two case reports]. / Die perioperative Therapie des Von-Willebrand-Syndroms Darstellung von Pathophysiologie, klinischer Problematik und Therapieoptionen anhand von 2 Fallberichten.

Von Willebrand disease (vWD) is the most widespread inherited bleeding disorder caused by quantitative or qualitative abnormalities of von Willebrand factor (vWF), an adhesive glycoprotein found in blood plasma and platelets and participating in primary and secondary/endothelium haemostasis as well. Although bleeding symptoms are often mild or moderate, patients with vWD represent a very heterogenous group with different phenotypes and a wide variability of the clinical pattern. In accordance with different defects of vWF, vWD is classified into various types and subtypes. This is illustrated by two case reports of patients with different types of vWD. Two main therapeutic options are available for the prevention and treatment of bleeding: desmopressin (DDAVP) and replacement therapy with plasma concentrates containing both factor VIII and vWF. DDAVP is the treatment of choice for most patients with type 1, representing about 80% of all patients with vWD. In patients with most types of type 2 and in all patients of type 3, DDAVP alone is ineffective or even contraindicated, and it is usually necessary to switch to plasma concentrates. Although treatment of vWD seems to be relatively simple in most cases, the exact diagnosis and phenotype characterization requires specialized or expert laboratory facilities. Furthermore, no reliable screening method for the diagnosis of vWD exists. Acquired vWD has similar clinical features and laboratory findings to the congenital forms and is mostly associated with lymphoproliferative or autoimmune disorders or neoplasia.

Evaluation of the haemocompatibility of titanium based biomaterials.

The increased use of metallic biomaterials in contact with blood e.g. for the application as coronary stents leads to the development of new biomaterials. The main requirements for stents are high flexibility, high cold deformability and sufficient mechanical strength (static and dynamic), which can be obtained by strain hardening, radio-opacity and haemocompatibility. In order to investigate the properties of the metallic biomaterials in contact with blood, a comparison of the haemocompatibility of newly developed materials with established materials has been performed. To evaluate haemocompatibility without the influence of the geometry of the material, spherical powders produced by rotating electrode process (REP) were used in a dynamic test system with full human blood under two different stress conditions. The high shear stress simulates the arterial and the low shear stress simulates the venous situation. The use of a dimensionless score point (SP) system where the parameters of the haemocompatibility are determined with and without a material exposition allows an objective comparison of the materials used.

Activation of plasma coagulation by retransfusion of unwashed drainage blood after hip joint arthroplasty: a prospective study.

Twelve patients undergoing cementless hip joint arthroplasty were retransfused with unwashed drainage blood collected postoperatively. Global coagulation parameters, coagulation factors (factor V:C, factor VIII:C, activated factor XII, and factor XIII) and markers of thrombin generation (F1+2 Fibrin split products, thrombin-antithrombin complexes), fibrin generation (fibrinogen and fibrin degradation products), and fibrinolysis (D-dimers, thrombin degradation products, plasminogen) were determined. High levels of factor XIIa, thrombin and fibrin generation markers, and markers of fibrinolysis were present in the shed blood. After retransfusion (mean, 433 mL), increased levels of these markers together with decreased values for factor XIII and plasminogen were indicative of renewed clot formation and fibrinolysis in the circulation. These changes were highly significant compared with preretransfusion values. The unwashed drainage blood contained high levels of procoagulation material and induced an activation of the plasma coagulation pathway with renewed clot formation and fibrinolysis in the patients.

Haemocompatibility of endovascular coronary stents: Wiktor GX.

The stent to be examined (Wiktor-Stent, Medtronic ESTC, Kerkrade, NL) was mounted into a closed-loop tubular-system and perfused with platelet-rich plasma (PRP). As controls the tubular-system without stent (as non-thrombogenic control) and secondly the tube filled with glassbeads (as thrombogenic control) were evaluated. A decrease in the number of singularly circulating thrombocytes correlated well with an increases in circulating platelet aggregates. The increasing activation of thrombocytes was demonstrated by immunolabelling of surface structures (CD 62) which become prominent on activation of thrombocytes. The increase in case of the non-thrombogenic controls is thought to be due to the action of the roller-pump. This increase was coincident with an increase in immunologically labelled GPIIb/IIIa receptors and well correlated with an increase in platelet activation as demonstrated by the elevated CD 62 label. In spite of the use of anticoagulation principles in the perfusion model, thrombin was generated (measured by the TAT-complex) in all three cases and the completed coagulation (measured by the occurrence of fibrin D-dimers) also happened. The amount of D-dimers was small, however, in the cases of non-thrombogenic controls and of tubes equipped with stents. Only after the contact of PRP with tubes filled with glass-beads a significant increase in D-dimers followed. In conclusion the implantation of a stent led to an activation, adherence and aggregation of thrombocytes to a somewhat greater extent as in the control-system. It has, however, a much less thrombogenic surface than glass-beads.

The polysaccharide fucoidan inhibits microvascular thrombus formation independently from P- and L-selectin function in vivo.

BACKGROUND: Adhesion molecules of the selectin family (mainly P- and L-selectin) have been suggested to mediate interactions between platelets, leukocytes and endothelial cells in thrombus formation. The polysaccharide fucoidan has anticoagulative properties, but is also able to bind and block the function of the selectins. Here, we investigated in vivo (i) if fucoidan can prevent microvascular thrombus formation, and (ii) whether this is potentially mediated by the inhibition of P-and/or L-selectin. MATERIALS AND METHODS: For this purpose, we used intravital microscopy in the mouse cremaster microcirculation in which thrombosis was induced photochemically by light exposure to individual arterioles and venules after intravenous (i.v.) injection of FITC-dextran. RESULTS: We found that intravenous administration of fucoidan significantly prolonged the time required for complete occlusion in arterioles and venules by almost seven- and nine-fold, respectively. In contrast, treatment with monoclonal antibodies against P- and L-selectin had no effect on the development of microvascular thrombosis. Fucoidan and also the anti-P-selectin antibody completely inhibited baseline venular leukocyte rolling in the cremaster muscle, indicating that these treatment regimes abolished P-selectin function. Importantly, fucoidan and the anti-P-selectin antibody had no effect on systemic platelet and leukocyte counts. On the other hand, we found that fucoidan treatment significantly altered coagulation parameters, including prothrombin time (Quick percentage), activated partial thromboplastin time (APTT) and thrombin clotting time (TCT), which may explain the potent in vivo anticoagulative effect of fucoidan observed here. CONCLUSIONS: Taken together, our novel findings suggest that fucoidan effectively prevents microvascular thrombus formation induced by endothelial damage in arterioles and venules in vivo. This protective effect of fucoidan is not attributable to inhibition of P- and L-selectin function but may instead be related to the anticoagulative capacity of fucoidan.