The interaction of platelets with von Willebrand factor is essential for primary hemostasis. Concentration and activity of plasma von Willebrand factor are routine parameters in the assessment of ...hemostasis disorders. In addition to plasma von Willebrand factor, platelet von Willebrand factor, synthesized in megakaryocytes and stored in α-granules of circulating platelets, is known to contribute to primary hemostasis and the microenvironment of thrombus formation. The laboratory assessment of platelet von Willebrand factor however is cumbersome and not widely established as a routine parameter. We here propose a method for laboratory assessment and reporting of platelet von Willebrand factor potentially useful for laboratory routines in specialized laboratories. Our model allows to describe platelet von Willebrand factor as 1. the concentration of platelet von Willebrand factor in whole blood, 2. the amount of platelet von Willebrand factor in a sample with a defined concentration of 1000 platelets/nl, and 3. the concentration of platelet von Willebrand factor in one platelet. According to our results in healthy individuals, the proportion of platelet von Willebrand factor activity is estimated to be about 10% of total von Willebrand factor in human plasma under physiological circumstances. The concentration of platelet von Willebrand factor is estimated to be 0.4 IU/ml in a sample with a defined concentration of 1000 platelets/nl and to be about 42 IU/ml in one platelet (both expressed as VWF:Ag).
Background: Recombinant von Willebrand factor (rVWF), which was licensed in the United States in 2015, has the multimeric distribution of freshly secreted VWF with ultralarge (UL) and high molecular ...weight multimers (HMWM) from endothelial cells and megakaryocytes since it has never been exposed to ADAMTS13 or any other proteolytic enzyme. Measurement of closure time (CT) using the platelet function analyzer-200 (PFA-200) is highly sensitive to the presence of UL VWF multimers in added VWF concentrates. The PFA-200 is fully automated and can be used as a reliable point-of-care method to evaluate primary hemostasis. Although it is sensitive to presence of UL VWF multimers, there could be significant clinical utility when used to monitor rVWF replacement therapy. The ability to monitor and optimize the dosing of rVWF contributes to patient safety, especially in situations where the bleeding and thrombotic risk needs to be carefully balanced (e.g., cardiac assist device).
Objective: The aim of this in-vitro study was to demonstrate the detectability of rVWF spiked in VWF-deficient blood from patients with severe von Willebrand disease (VWD) with quantitative and functional pathologies using a functional testing device. We hypothesized that (1) whole blood samples from VWD patients spiked with rVWF would show a normalization in PFA-CT and (2) that a dose-response relationship could be demonstrated.
Methods and results: We selected 12 patients diagnosed with VWD from our database. A therapeutic dose of rVWF product (1 IU/ml) was spiked in VWD patients´ whole blood samples and PFA-CTs were measured. Furthermore, we investigated PFA-CTs under incremental doses of rVWF (0.1, 0.2, and 0.5 IU/ml). The PFA-CTs were normalized in VWD patients´ whole blood samples spiked with rVWF. Additionally, incremental doses of rVWF resulted in a progressive and dose-dependent PFA-CT correction.
Conclusion: Our in-vitro data indicate that the PFA-200 is a useful tool to detect rVWF. As the PFA-CT correction is dose dependent, the rVWF might be reliably monitored with a point-of-care analytical method during replacement therapy.
Activation of the platelet Fc-receptor CD32a (FcγRIIA) is an early and crucial step in the pathogenesis of heparin-induced thrombocytopenia type II (HIT) that has not been therapeutically targeted. ...Downstream FcγRIIA Bruton tyrosine kinase (BTK) is activated; however, its role in Fc receptor–induced platelet activation is unknown. We explored the potential to prevent FcγRIIA-induced platelet activation by BTK inhibitors (BTKi's) approved (ibrutinib, acalabrutinib) or in clinical trials (zanubrutinib BGB-3111 and tirabrutinib ONO/GS-4059) for B-cell malignancies, or in trials for autoimmune diseases (evobrutinib, fenebrutinib GDC-0853). We found that all BTKi's blocked platelet activation in blood after FcγRIIA stimulation by antibody-mediated cross-linking (inducing platelet aggregation and secretion) or anti-CD9 antibody (inducing platelet aggregation only). The concentrations that inhibit 50% (IC50) of FcγRIIA cross-linking–induced platelet aggregation were for the irreversible BTKi's ibrutinib 0.08 µM, zanubrutinib 0.11 µM, acalabrutinib 0.38 µM, tirabrutinib 0.42 µM, evobrutinib 1.13 µM, and for the reversible BTKi fenebrutinib 0.011 µM. IC50 values for ibrutinib and acalabrutinib were four- to fivefold lower than the drug plasma concentrations in patients treated for B-cell malignancies. The BTKi's also suppressed adenosine triphosphate secretion, P-selectin expression, and platelet-neutrophil complex formation after FcγRIIA cross-linking. Moreover, platelet aggregation in donor blood stimulated by sera from HIT patients was blocked by BTKi's. A single oral intake of ibrutinib (280 mg) was sufficient for a rapid and sustained suppression of platelet FcγRIIA activation. Platelet aggregation by adenosine 5′-diphosphate, arachidonic acid, or thrombin receptor-activating peptide was not inhibited. Thus, irreversible and reversible BTKi's potently inhibit platelet activation by FcγRIIA in blood. This new rationale deserves testing in patients with HIT.
•Six different BTKi's blocked platelet activation in blood after FcγRIIA stimulation by cross-linking, anti-CD9 antibodies, or HIT serum.•Established oral irreversible and novel reversible BTKi's may offer a new option to treat HIT.
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Abstract Introduction The paradigm of activation induced platelet aggregation has recently been refuted under blood flow conditions with shear rates exceeding 20,000 s - 1 . These lead to reversible ...rolling platelet aggregates, which were dependent on the presence of immobilized and soluble von Willebrand factor. Material and Methods In vitro experiments using direct fluorescence video-microscopy were performed in wall parallel and stagnation point flow chambers with shear rates raised from 20,000 to 50,000 s - 1 . Washed blood cell suspension containing recombinant von Willebrand factor (rVWF) was perfused over rVWF or collagen coated surfaces. Results Here we show for the first time with the visualization of rVWF that not only colloid and polymer, i.e. platelets and VWF, form a composite, but that VWF itself is capable of entirely reversible self-assembly. On a collagen surface the platelet-VWF-conglomerates did not roll but VWF nets bound permanently to the collagen fibers and captured and immobilized platelets from the flow. Lowering the shear rate below the threshold of 20,000 s - 1 no longer dissolved these deposits. Ultralarge multimer containing rVWF was most effective compared to normal sized rVWF. The presence of ADAMTS13 limited rolling aggregate and platelet-VWF-conglomerate formation to a time window of 7-8 minutes. Changing wall parallel flow to stagnation point flow halved the required shear rate threshold. Conclusion We conclude that flow dynamics can trigger reversible von Willebrand factor self-assembly and platelet-VWF-conglomerate accrual, which are regulated by ADAMTS13 to a time span needed by coagulation to stabilize it, e.g. in case of vessel injury.
When using artificial surfaces that come into contact with the bloodstream, it is important to consider the undesirable consequences of thrombus formation and embolization. Although great progress ...has been made by creating new surfaces and antithrombotic coatings or evaluating flow conditions, unexpected platelet adhesion and aggregation can lead to the sudden formation of an adverse thrombus. Our experiments in a stagnation point flow chamber with citrate-anticoagulated whole blood and ADP-stimulated platelets mimicked the situations of implanted artificial organs, e.g., mechanical circulatory support devices, or extravascular circulation. With video microscopy, real-time platelet characteristics were observed at shear rate levels between 50 and 500 s
−1
on glass, von Willebrand factor, and polyurethane surfaces for at least 5 min after the first contact. Platelet adhesion and aggregation were observed with distinctness in aggregate size, surface coverage, aggregate size, probability of an embolic event, and platelet contraction. The probability of an embolic event increased at lower flow rates. Additionally, platelet contraction was affected by the flow rate. Raising the flow rate intensified the platelet contraction. With this setup, the microembolization caused by surface contact and flow and platelet contraction can be detected in a real-time direct observation. This capability addresses both technical and clinical issues, such as thrombus and embolus formation, and may improve the research on the hemocompatibility of biomaterials.
Ultralarge multimers (ULM) of VWF are considered to be the most active with respect to binding to platelets and to subendothelial structures and therefore are of critical importance for the function ...of VWF in stabilizing the primary hemostatic plug. In contrast to plasma-derived FVIII-VWF concentrates, human rVWF obtained from mammalian cell culture retains the full-spectrum of intact multimers, including ULM, as physiologically formed in the Golgi apparatus and stored in platelet α-granules and endothelial cell Weibel-Palade bodies. In the course of physico and biochemical, functional and animal studies, rVWF exhibited superiority in structure and function compared to pdVWF. These effects seemed to correlate with the multimer size and therefore might be attributed to the presence of ULM in rVWF preparations. The pharmacokinetic (PK), safety and efficacy characteristics seen in preclinical studies were further demonstrated in clinical trials.
Short-term physical training in bronchial asthma Bundgaard, A; Ingemann-Hansen, T; Halkjaer-Kristensen, J ...
British journal of diseases of the chest,
01/1983, Letnik:
77, Številka:
2
Journal Article
The effect of two types of physical training on patients with perennial asthma were compared in a blind, controlled, randomized study. Eleven of 27 adults with asthma performed a physical training ...programme which did not change their oxygen consumption (control group). The remaining 16 asthmatics performed a physical training programme which improved their maximal oxygen consumption (training group). Both of the training programmes were performed for 1 hour, twice a week during a period of 2 months. No complications were reported during the performance of the training programmes. The doses of all medicines apart from beta 2-agonist aerosol were unchanged during the training period. The patients inhaled beta 2-agonist aerosol if their peak expiratory flow (PEF) was less than 60% of their maximal PEF. The training group decreased their use of aerosol from an average of 4.94 puffs per day to 3.41 puffs per day (P less than 0.05). The control group did not change their use of beta 2-agonist aerosol significantly. It is concluded that physical exercise which improves the maximal oxygen consumption decreases the use of beta 2-agonist spray and that heavy exercise is well tolerated by asthmatics.
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