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Activation and destruction of platelets in patients with rheumatic heart disease.

We evaluated the activation and destruction of platelets in 24 patients with rheumatic heart disease (RHD) involving the mitral valve. Ex vivo platelet aggregation induced by adenosine diphosphate (ADP) and collagen was significantly increased in RHD patients as compared with normal controls. Plasma levels of beta-thromboglobulin (beta-TG) and platelet factor 4 were also elevated. Plasma concentrations of glycocalicin, a proteolytic fragment of platelet membrane glycoprotein Ib, were increased, while platelet counts were decreased in RHD patients as compared with normal controls. RHD patients with, versus those without, atrial fibrillation demonstrated significant differences in beta-TG levels and platelet counts. However, we observed no difference in glycocalicin levels between the two groups. The present study demonstrated that increased platelet activation, as well as platelet destruction, occur in patients with RHD.

Adult↗

Xenon does not affect human platelet function in vitro.

We sought to determine whether xenon affects platelet glycoprotein expression and platelet-related hemostasis in vitro at a clinically relevant concentration. Human whole blood was stimulated with either adenosine diphosphate or the thrombin receptor agonist peptide (TRAP)-6 after incubation with 65% xenon. Halothane at 2 minimum alveolar anesthetic concentration was used as a positive control. Platelet function and activation were evaluated with two-color flow cytometry. The expression of the platelet glycoproteins GPIIb/IIIa, GPIb, and P selectin were detected with fluorochrome-conjugated monoclonal antibodies. In vitro measurement of platelet-related hemostasis under conditions of high shear stress was performed in citrated whole blood with a platelet function analyzer (PFA-100((R))) by using collagen/epinephrine and collagen/adenosine diphosphate cartridges. Xenon did not affect basal or agonist-induced expression of platelet membrane glycoproteins, activation-dependent conformational changes of the GPIIb/IIIa receptor, expression of P selectin, or PFA closure times. In contrast, halothane reduced TRAP-6-induced activation of the GPIIb/IIIa complex. Furthermore, collagen/epinephrine-induced PFA closure time was significantly prolonged. These results demonstrate that xenon does not affect the unstimulated or agonist-induced platelet glycoprotein expression, activation of GPIIb/IIIa, or platelet-related hemostasis.

Anesthetics, Inhalation↗

Safety and antiplatelet effect of murine monoclonal antibody 7E3 Fab directed against platelet glycoprotein IIb/IIIa in patients undergoing elective coronary angioplasty.

BACKGROUND: Excessive platelet deposition at the site of arterial damage due to coronary angioplasty plays an important role in the pathophysiology of both abrupt closure and restenosis after that procedure. Even aspirin, a relatively weak platelet antagonist, decreases the complications of coronary angioplasty. This study was designed to provide preliminary safety and efficacy data on the use of a much more powerful antiplatelet agent, m7E3 Fab, a prototype murine monoclonal antibody fragment that binds directly to the platelet glycoprotein IIb/IIIa receptor mediating aggregation in patients undergoing coronary angioplasty. METHODS: Twenty-three patients referred for elective coronary angioplasty who met prespecified criteria designed to minimize risk of bleeding received, in groups of four to six patients, escalating bolus doses of 0.15 to 0.35 mg/kg of this agent immediately before coronary angioplasty. Heparin was administered in the usual manner, but aspirin was withheld for 24 hours before coronary angioplasty and until bleeding times and platelet aggregation had returned to normal after angioplasty. Glycoprotein IIb/IIIa binding site occupation, platelet aggregation response to 20 microM of adenosine diphosphate, and data on bleeding times were acquired at baseline and at 2, 6, 24, 48 and 72 hours after m7E3 Fab administration. Clinical safety and efficacy were also monitored throughout the time of hospitalization, and delayed antimurine immune responses were assayed. Five similar patients received aspirin (325 mg orally per day) but otherwise received the same treatment, thus serving as controls. RESULTS: Treatment with m7E3 Fab resulted in a dose-dependent occupation of binding sites to a maximum of 93% at 2 hours in the highest-dose groups, with an associated graded inhibition of platelet aggregation and increase in bleeding time significantly exceeding that seen in control patients, with a gradual recovery over 6 to 48 hours. Percutaneous transluminal coronary angioplasty was successfully performed in 18 of 21 patients (86%) in whom it was attempted. Arterial and venous sheath removal 24 hours after m7E3 Fab dosing was largely uneventful. No thrombotic complications were seen, and only one patient (excluding a patient who underwent uneventful urgent bypass surgery) had bleeding severe enough to require packed red cell transfusion. Eight patients (36%) developed late antibody titers against m7E3 Fab. CONCLUSIONS: This murine monoclonal antibody provides potent antiaggregatory action and thus may be useful in preventing thrombotic complications of coronary angioplasty, but studies of its safety and efficacy during longer infusions and with larger numbers of patients are needed. Less immunogenic forms of the antibody may be more clinically useful.

Angioplasty, Balloon, Coronary↗

In vitro effects of poly-N-acetyl glucosamine on the activation of platelets in platelet-rich plasma with and without red blood cells.

BACKGROUND: This study was performed to assess the effect of poly-N-acetyl glucosamine fiber slurry on plasma clotting proteins, platelets, and red blood cells in the clotting of the blood. METHODS: Citrate phosphate dextrose whole blood was stored at 22degreesC for 48 hours to prepare platelet-poor plasma, platelet-rich plasma (PRP), and PRP plus red blood cells with hematocrit values of 20%, 35%, and 45% with and without an equal volume of poly-N-acetyl glucosamine fibers (1 mg/mL 0.9% NaCl). RESULTS: Thromboelastogram data show that poly-N-acetyl glucosamine fibers (p-GlcNAc) significantly reduced the R time in platelet-poor plasma, PRP, and PRP supplemented with red blood cells. Poly-N-acetyl glucosamine fibers increased, but not significantly, Annexin V and factor X binding to platelets, platelet microparticles, and red blood cell Annexin V binding. Poly-N-acetyl glucosamine fibers increased the production of thromboxane B2 by PRP. CONCLUSION: Poly-N-acetyl glucosamine slurry activates platelets.

Acetylglucosamine↗

Implementation of the INTERCEPT Blood System for Platelets into routine blood bank manufacturing procedures: evaluation of apheresis platelets.

BACKGROUND AND OBJECTIVES: The INTERCEPT Blood System for Platelets utilizes amotosalen-HCl (S-59) in combination with ultraviolet A (UVA) light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet concentrates (PCs). To facilitate implementation of this technique into routine blood bank manufacturing procedures, this study evaluated the impact of different time settings of photochemical treatment on in vitro platelet function. MATERIALS AND METHODS: Platelets derived from apheresis (6.5-7.0 x 10(11) platelets) were resuspended in 240 ml of autologous plasma and 360 ml of platelet additive solution (PAS III) and split into two equal-sized PC units. Whereas one unit was not treated, the other was treated with 150 microm amotosalen and 3 J/cm2 UVA light followed by a compound adsorption device (CAD) step for reduction of residual amotosalen and photoproducts. In a first series of experiments (arm A, n = 7), PC units were photochemically treated after an overnight storage period of 16-23 h followed by a CAD step of 4 h. In a second series (arm B, n = 8), photochemical treatment occurred after a short storage time of 4 h with a subsequent CAD step of 16 h. Platelet function was evaluated by assaying blood gas analysis, glucose and lactate concentration, lactate dehydrogenase (LDH), hypotonic shock response (HSR) and the expression of CD62p, over a period of 7 days. RESULTS: Neither of the photochemical treatment procedures showed differences for pH, pCO2, pO2, HCO3, glucose consumption or platelet activation until the end of day 7. Increased lactate values detected for the treated units of arm A at the end of the storage period were independent from the PCT time setting. CONCLUSIONS: Photochemical pathogen inactivation with different initial resting periods between 4 and 23 h, and different CAD steps of 4 and 16 h, had no influence on the platelet in vitro function during 7 days of storage.

Adsorption↗

Impact of tobacco smoking on platelet function in apheresis products in vitro.

BACKGROUND AND OBJECTIVES: The aim of this study was to analyse the platelet function, over a 5-day time-period, of apheresis-derived platelet concentrates obtained from smokers and non-smokers. MATERIALS AND METHODS: Smoker and non-smoker plateletpheresis products were investigated on days 1, 3 and 5 of storage. Receptor expression (as evaluated by flow cytometry) and the platelet aggregation response were measured. RESULTS: There was only a slight loss of platelet function in apheresis products from smokers compared to non-smokers. CONCLUSIONS: Smoking does not significantly change the quality of platelet preparations. The current practice, of not to exclude smokers from platelet donation, can be continued.

Adenosine Diphosphate↗

In vitro effects of gelatin solutions on platelet function: a comparison with hydroxyethyl starch solutions.

The aim of this study was to investigate the effects of various gelatin and hydroxyethyl starch solutions on platelet reactivity. Citrated whole blood was obtained from 20 healthy volunteers. Expression of glycoprotein (GP) IIb-IIIa and p-selectin were determined using whole blood flow cytometry on both resting and agonist-activated platelets before and after in vitro haemodilution (20% and 40%) using oxypolygelatin, modified gelatin, urea-linked gelatin, hydroxyethyl starch (HES) 130 (mean molecular weight in kDa), HES 200, HES 450 and HES 550. High degrees of haemodilution using oxypolygelatin had no significant effect, similar to HES 130, whereas modified gelatin inhibited GP IIb-IIIa expression, similar to HES 200 and HES 450. Urea-linked gelatin significantly increased the expression of GP IIb-IIIa, similar to HES 550. p-selectin expression remained unchanged in all samples. The present in vitro study indicates that chemical characteristics of colloidal solutions modulate their influence on platelet reactivity.

Adult↗

Decreased stability and structural heterogeneity of the residual platelet glycoprotein IIb/IIIa complex in a variant of Glanzmann's thrombasthenia.

A patient is described with a disturbance of platelet function comparable to that in Glanzmann's thrombasthenia. Platelet aggregation and binding of fibrinogen to the patient's platelets were defective and thrombin-induced clot retraction was absent. The platelet fibrinogen content was only moderately reduced. As measured by monoclonal antibody binding in the presence of divalent cations, the platelets contained about 15% of the normal amount of GPIIb and GPIIIa and only 6% of the normal amount of intact GPIIb/IIIa complex. The residual GPIIb/IIIa complex exhibited a decreased stability as shown by the lack of binding of a complex-dependent anti-GPIIb/IIIa antibody to platelets incubated with ethylene diamine tetraacetic acid (EDTA) at 22 degrees C. Crossed immunoelectrophoresis (CIE) in the presence of divalent cations showed partial dissociation of GPIIb/IIIa as well as the presence of two forms of the residual intact GPIIb/IIIa complex. In addition, both CIE in the presence of the EDTA and two-dimensional sodium dodecyl sulphate (SDS) gel electrophoresis showed the presence of two forms of GPIIb. This form of thrombasthenia is characterized by a defective platelet function, a marked reduction of GPIIb and GPIIIa, decreased stability of the residual GPIIb/IIIa complex and structural heterogeneity of GPIIb.

Adult↗

Inhibition and reversal of platelet aggregation by alphaIIbbeta3 antagonists depends on the anticoagulant and flow conditions: differential effects of Abciximab and Lamifiban.

Shear influences platelet aggregate formation and stability, as well as the inhibitory capacities of antithrombotic drugs. We compared the inhibitory and disaggregating properties of two distinct alphaIIbbeta3 antagonists, Abciximab and Lamifiban, on platelet aggregation induced by adenosine diphosphate (ADP) (5 micromol/l) in platelet-rich plasma (PRP), in an aggregometer (poorly defined low shear, <100/s) and in a microcouette at arterial shear rate (1,000/s). Platelet aggregation was detected by changes in light transmission in the aggregometer (TA), and by particle counting with a flow cytometer (PA). Lamifiban (1 mumol/l) completely inhibited TA or PA induced by ADP in citrated PRP in the aggregometer or microcouette. In contrast, Abciximab (2 micromol/l) only partially inhibited PA in the microcouette while blocking both TA and PA in the aggregometer. Moreover, Abciximab did not reverse platelet aggregates formed either in the microcouette or in the aggregometer, whereas Lamifiban caused complete reversal. On the contrary, Abciximab completely inhibited platelet aggregation induced by ADP in hirudin/d-Phe-Pro-Arg-chloromethylketone PRP in the microcouette. Our results demonstrate a marked dependence of inhibitory capacity of Abciximab on shear conditions, with citrate anticoagulant responsible for the residual aggregation, in contrast to Lamifiban, another alphaIIbbeta3 antagonist interacting with a distinct site on beta3.

Abciximab↗

Quality of platelet concentrates irradiated with UVB light: effect of UV dose and dose rate on glycocalicin release and correlation with other markers of the platelet storage lesion.

The amount of membrane-associated glycoprotein Ib in platelet concentrates (PCs) irradiated with a high dose of UVB light has been shown to be significantly reduced after 48 h storage. We recently corroborated this finding when we noted an increase in the supernatant levels of glycocalicin (GC, a major segment of glycoprotein Ib) in UVB-treated PCs during storage. The aim of the present study was to determine whether GC release was related to both the UV dose and the rate of dose delivery. Plateletpheresis concentrates obtained from five donors were pooled and split into five equal parts. Four of these were treated with 7500 and 15,000 mJ/cm2 UVB using two prototype UV sources with differing rates of dose delivery; namely, Baxter (BAT) and British Aerospace (BAC) cabinets, with the latter having the slower rate of delivery. On days 1 and 5 of storage, GC levels in the supernatants of PCs were determined by ELISA. Moreover, the following parameters were also assessed: platelet and WBC count; hypotonic shock response (HSR) and platelet aggregation response to ADP, ADP+collagen, ADP+arachidonic acid and ristocetin; pH; supernatant levels of lactate, glucose, von Willebrand factor (vWf) and beta-thromboglobulin (beta TG). The results revealed an association of GC release with UVB dose using both UV sources, although this was more apparent in the BAC system, in which glycocalicin release at day 5 of storage was as follows (microgram/ml, mean +/- SD): 4.8 +/- 0.3 and 9.5 +/- 3.6 at 7500 and 15,000 mJ/cm2 respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Platelets↗

5-day storage of platelet concentrates in CLX containers: effect of type of agitation.

To determine the degree of platelet damage produced by different modes of agitation during storage of concentrates for 5 days in CLX blood bags, we studied pH, platelet counts, release of LDH and beta thromboglobulin, morphology and osmotic recovery. Platelets were maintained at 20-24 degrees C on elliptical, 6-rpm circular, 2-rpm circular and flat bed agitators. At 72-120 h platelet concentrates stored on the flat bed shaker had significantly lower pH values than units stored on the elliptical or on either of the circular rotators (p less than 0.05). The percent LDH discharged was highest for the units stored on the elliptical rotator (p less than 0.05). Remaining tests of platelet function were not significantly different for concentrates stored on any of the four agitators. Flat bed shakers were unable to resuspend the platelet 'button' which formed after the final preparative centrifugation. Based on our in vitro studies, we conclude that due to problems with low pH values, flat bed shakers may not be optimal for storing platelet concentrates in CLX blood bags and that some other form of agitation should be used.

Blood Platelets↗

Platelet antibody binding and spontaneous aggregation in 21 lupus anticoagulant patients.

In order to ascertain the role of the antiphospholipid antibody in the pathogenesis of thrombotic disorders, a study of 21 lupus anticoagulant-positive patients was done by a number of serologic and functional platelet tests. In immunofluorescent studies, we found that 80% showed a mitochondrial pattern on HEp cells and all patients gave an ubiquitously intense staining of donor platelets. By a microscopic spontaneous aggregation test, all lupus anticoagulant-positive patients showed accelerated platelet aggregation which was calcium-independent. The results show that the plasma of lupus anticoagulant-positive patients contains a platelet-binding antibody and causes spontaneous agglutination of platelets. These properties may play a role in the thrombogenesis common to the antiphospholipid syndrome.

Antiphospholipid Syndrome↗

Role of lactate in platelet storage lesion.

It is known that lactate accumulation may cause a pH fall in platelet concentrates (PC) during storage, and this phenomenon causes platelet morphological lesions and loss of platelet in vivo viability. In this study, we added increasing amounts of lactate to identical PC in order to evaluate the role of hydrogen ion accumulation in determining platelet activation and lesion during storage. Six hours after PC preparation, lactate was added to PC1 and PC2 at 20 and 12 mM final concentrations, respectively, while PC3 served as control. In PC1, pH was lower than 6.3, and platelet function and discoid morphology were lost. PC2 were stored for 7 days at pH values ranging from 6.4 to 6.6, and most results of in vitro measurements reflecting platelet function such as osmotic reversal, ATP release and aggregation in response to different stimuli were not significantly inferior when compared to controls. The addition of lactate had no apparent effect on the rise of platelet activation markers P-Selectin, lysosome-like protein gp 53, platelet-bound fibrinogen and granulophysin, while a reduction of borderline significance was observed in glycoprotein Ib expression after pH reduction to values lower than 6.6. It is concluded that the rise of platelet activation markers during storage reflects platelet lesions different from those determined by lactate per se.

Blood Platelets↗

In vitro and in vivo properties of various types of platelets.

The most popular procedures used to prepare platelet concentrates (PC) can be categorized into methods using platelet rich plasma (PRP) obtained by soft centrifugation of whole blood units, methods using buffy coats (BC) obtained by hard centrifugation of whole blood units, and apheresis procedures. The main feature that differentiates apheresis PC from whole blood unit derived PC is that apheresis reduces the number of donors necessary to support a recipient, although the advantages related to this feature have not been conclusively documented. From the biochemical point of view, a limited number of comparative studies and a large series of non-comparative studies indicate that differences in PC obtained with different protocols depend more on the performances of the production laboratory rather than the preparative approach--PRP, BC or apheresis--itself. Similarly, the clinical effectiveness of PC seems to depend more on PC age, storage modalities and recipient's conditions rather than primarily on the method of PC preparation. Despite the basic differences in the three preparative approaches, all methods share a number of key elements that are necessary pre-requisites for a successful platelet transfusion therapy: minimization of the risk of bacterial contamination by careful donor skin disinfection and temperature control during PC production and storage; protection of aerobic metabolism during PC storage; laboratory quality control including at least pH and volume determinations, platelet and white cell counts and visual inspection of the swirling phenomenon at time of release. Future directions in the field of PC production include the development of new products such as infusible platelet membranes, thrombospheres, thromboerythrocytes and reconstituted freeze-dried platelets.

Adult↗

In vitro function of platelet concentrates prepared after filtration of whole blood or buffy coat pools.

BACKGROUND/METHOD: Data on the quality of platelet concentrates (PC) produced by the buffy coat method and stored beyond 5 days in plasma are limited. We therefore evaluated the quality of PCs prepared by leucocyte depletion of whole blood (Terumo WBSP, n = 10) or a buffy coat pool (Pall Autostop, n = 10), and stored for 7 days in plasma by assessing platelet parameters and markers of platelet activation. RESULTS: In both types of PC, levels of glucose decreased during storage but were not totally depleted (> 11 mM on day 7). In contrast, lactate levels increased on storage and was consistently < 20 mM throughout, with pH maintained at > 6.8 in all units. Hypotonic shock response scores were > 47% in all units at day 7. On day 1, markers of platelet activation were significantly higher in WBSP PC, but by day 7 were similar for percentage CD63+ and CD62P + (40%) with levels of platelet microparticles and annexin V binding two-fold higher in WBSP. The expression of CD61 did not alter during storage and the percentage of platelets expressing CD42b was > 88% in all units on day 7. RANTES (Regulated on activation, normal, T-cell expressed and secreted) and TGFbeta released from platelets by day 7 was < 800 ng/ml and 90 ng/ml, respectively. C3a(desarg) increased throughout storage in both types of PC, but without a commensurate increase in the terminal complex SC5b-9 or activation of factor XII. CONCLUSION: Our data indicates that the in vitro characteristics of PCs prepared using these methods is maintained over storage for 7 days in plasma and is not associated with significant deterioration of platelet function. ONE SENTENCE SUMMARY: In vitro function of platelet concentrates prepared by either filtration of whole blood, or pooled buffy coats.

Analysis of Variance↗