Comparison of coated-platelet levels in patients with essential thrombocythemia with and without hydroxyurea treatment.

Essential thrombocythemia (ET) is an acquired myeloproliferative disorder with sustained increase of platelet count. This disease may be associated with thrombotic or bleeding complications due to the altered number and function of platelets. Coated-platelets produced by a simultaneous activation of collagen and thrombin represent a subpopulation of activated platelets with high prothombinase activity and the retention of several α-granule-derived coagulation factors on their surface. There is a growing body of evidence for a relationship between variable levels of coated-platelets and different hemostatic alterations. However, no data are available on coated-platelet formation in the pathogenesis of ET in the presence or absence of treatment. The levels of coated-platelets in 43 ET patients (15 non-treated and 28 hydroxyurea-treated) without known thrombotic or hemorrhagic complications were analyzed using flow cytometry. These results were compared with data of 31 healthy individuals. In addition, platelet function was analyzed with PFA-100 analysis, and P-selectin (CD62) positivity was also measured by flow cytometry. Increased P-selectin expression was detected with prolonged PFA-100 closure times in the ET group; however, significantly lower levels of coated-platelets were found in non-treated ET patients compared to controls (23.1 ± 8.8% vs. 37.6 ± 12.7%, p = 0.0008). This tendency was more evident in patients with JAK2-V617F mutation. Patients on hydroxyurea treatment had elevated coated-platelet levels (34.1 ± 12.3%) close to the normal value. In conclusion, lower than normal levels of coated-platelets were generated in ET, which were significantly (p = 0.0008) increased by hydroxyurea treatment. We suppose that abnormal coated-platelet level may also contribute to platelet dysfunction in ET.

Click reaction synthesis of carbohydrate derivatives from ristocetin aglycon with antibacterial and antiviral activity.

New sugar derivatives of ristocetin were prepared by copper-catalyzed 1,3-dipolar cycloaddition reaction using azido-ristocetin aglycon and various propargyl glycosides. Some of them were found to be active against gram-positive bacteria and showed favorable antiviral activity against the H1N1 subtype of influenza A virus.

Increased platelet glycoprotein Ib receptor number, enhanced platelet adhesion and severe cerebral ischaemia in a patient with polycythaemia vera.

The present study describes severe multiplex cerebral ischaemic laesions in a male patient being diagnosed with polycythaemia vera (PV). In contrast to previous publications, unique platelet receptor pattern with normal platelet count was identified. Glycoprotein Ib receptor number on the surface of resting platelets was increased two-fold and almost three-fold in case of activated platelets compared to the controls. More over, in an in vitro study when whole blood was circulated both at venous and arterial shear conditions and shear rate was adjusted according to the blood viscosity, platelet aggregate/thrombus formation was characteristic on surfaces covered with purified von Willebrand factor while in case of controls the surface was covered with single platelets or platelet monolayer. Similar results with pathological findings have not been published in PV until now. Our result undersigns the necessity of antiplatelet therapy of PV patients, even at normal platelet count.

[Autologous bone marrow-derived stem cell therapy in patients with severe peripheral arterial disorder]. / Autológ csontvelôi eredetû ôssejtterápia eredménye elôrehaladott perifériás artériás érbetegségben.

BACKGROUND AND AIMS: Amputation is the only current option for relief of rest pain or gangrene in patients with severe peripheral arterial disease. Up to now, no effective blood-flow enhancement therapies are available. Autologous bone marrow-derived stem cell transplantation is an arising therapy modality with an option of building new blood vessels through endothelial stem and/or progenitor cells. PATIENTS AND METHODS: Five patients with severe peripheral arterial disorder were treated by autologous bone marrow-derived stem cell therapy. CD34+, CD133+ and CD45+/- cell number and ratio were determined. CD34+ cells were isolated by magnetic separation and collected into a 10 ml sample. The cell suspension was administered by local intramuscular injections (0.5-1.0 ml injections in the musculus gastrocnemius). The follow-up (before; 1, 3, 6, 9 and 12 months after the autologous bone marrow-derived stem cell therapy) based on clinical (rest pain, walking distance without pain, changes of non-healing ischaemic ulcers, ankle-brachial index) and laboratory (angiography, Color- and Laser-Doppler scan, measurement of transcutaneous oxygen tension and endothelial function test) parameters was documented and analyzed. RESULTS: Improvement of pain and walking distance was observed in all five cases. In three cases the non-healing ischaemic ulcers disappeared, in one other case they became smaller and thinner, and in one case no change was realized. The average of ankle-brachial index improved significantly (before: 0.41, twelve months after: 0.83). New collaterals were detected by angiography in three patients, but duplex ultrasonography detected improvement in one patient only. Before and 1, 6 and 12 months after stem cell therapy the transcutaneous oxygen tension changed on the foot from 18.80/16.78/23.83/37.50 mmHg, and on the calf from 36.66/31.25/45.00/37.30 mmHg. The macro- and microcirculation parameters did not show improvement after 1 month, however, after 3, 6, 9 and 12 months improved parameters were recorded. Severe adverse events were not observed. In one case elevated level of serum creatinine phosphokinase, and in another case a mild form of vasculitis were detected. CONCLUSION: autologous bone marrow-derived stem cell therapy with isolated CD34+ cells is effective, safe and results in sustained clinical benefit for patients with severe peripheral arterial disease.

Direct thrombin inhibitors and factor xa inhibitors can influence the diluted prothrombin time used as the initial screen for lupus anticoagulant.

CONTEXT: Lupus anticoagulant (LA) is a heterogeneous group of antiphospholipid antibodies. Among others, diluted prothrombin time (dPT) is a sensitive screening test for LA; however, the interpretation of LA tests is difficult in patients treated with anticoagulants. The effect of different types of anticoagulants on the result of LA tests, particularly on dPT, has not been studied extensively. OBJECTIVE: To determine whether the direct thrombin inhibitors lepirudin and argatroban and the predominantly factor Xa inhibitors enoxaparin, danaparoid, and fondaparinux could interfere with LA screening based on dPT. DESIGN: Each drug was added to normal and LA-positive plasmas in clinically relevant concentrations. Each sample was tested for dPT. Samples with factor Xa inhibitors were investigated before and after addition of heparinase. Mixing and confirmatory tests for LA were not performed. RESULTS: In the presence of lepirudin or argatroban, dPT increased notably and the dPT ratio exceeded the cutoff value even at subtherapeutic concentrations resulting in false positivity. With increasing factor Xa inhibitor concentrations, a linear increase of dPT ratios and false-positive results were also demonstrated. Although heparinase could almost completely neutralize the anti-Xa effect of all investigated factor Xa inhibitors, dPT ratio returned to the basal level only in case of enoxaparin. CONCLUSIONS: Here we provide evidence that both the direct thrombin and indirect factor Xa inhibitors influence dPT assay for LA, causing false positivity. This should be considered when interpreting LA results during anticoagulant therapy. However, dPT seems to be a reliable test for LA screening under enoxaparin therapy after neutralization by heparinase.

Stem cell therapy: a promising and prospective approach in the treatment of patients with severe Buerger's disease.

No effective blood-flow enhancement therapies are available for patients with severe peripheral arterial disease (SPAD), thus amputation remains the only option for relief of rest pain or gangrene. Autologous bone marrow-derived stem cell therapy (ABMSCT) is an emerging modality to induce angiogenesis from endothelial progenitors. A total of 5 patients with SPAD were treated by ABMSCT using isolated CD34+ cells with characterized phenotype administered by intramuscular injections. The follow-up before and 1, 3, 6, 9, and 12 months after ABMSCT was based on clinical (rest pain, walking distance without pain, nonhealing ulcers, ankle-brachial index [ABI]) and laboratory (angiography, duplex and laser ultrasonography, TcPO(2)) parameters. Significant improvement of pain and walking distance was observed in all patients. Nonhealing ulcers disappeared in 3 patients and became smaller and thinner in 1 patient. The average of ABI improved significantly on the treated limb but did not change on the contralateral limb. New collaterals were detected by angiography in 3 patients, but duplex ultrasonography detected improvement in one patient only. Laser ultrasonography showed a mild significant change, TcPO(2) values improved mainly on the foot. Severe adverse events were not observed. We conclude that ABMSCT with isolated CD34+ cells is safe, effective, and results in sustained clinical benefit for patients with SPAD.

Differential platelet deposition onto collagen in cone-and-plate and parallel plate flow chambers.

To routinely test the formation of thrombi and the effect of drugs modifying it, proper test systems are needed. Their design should rely on the laws of rheology and the physiology of laminar flow. To best model physiological or pathological shear conditions, parallel/linear and rotational type flow chambers are developed. We have compared the initial phase of platelet thrombus formation in a parallel plate flow chamber (PPC) and a cone-and-plate chamber (CPC) under von Willebrand dependent shear conditions. Blood was allowed to flow through human collagen type III surfaces at a shear rate of 1000 s(-1) for 150 s. Thrombus deposition was characterized by surface coverage, average area and height of thrombi. VWF distribution within thrombi was analyzed with confocal laser scanning microscopy. Reduced surface-specific platelet adhesion and aggregation (surface coverage and average thrombus size) were observed in CPC along with a significant increase in single platelet disappearance from the circulating blood. Our data suggest that the higher rate of platelet consumption in this device, as opposed to PPC, is limiting the adhesion to the surface. Consequently, surface-specific processes and aggregation in the flowing blood are both assessed using CPC, while comprehensive evaluation of surface-specific processes is best achieved with PPC. Therefore, the choice of chamber type as a diagnostic tool is purpose-dependent.