Addition Of Platelet-poor Plasma Research Articles

In vitro platelet aggregation defects in patients with myeloproliferative disorders and high platelet counts: Are they laboratory artefacts?

It has been recently shown that in vitro platelet aggregation is inhibited when platelet concentration in platelet-rich plasma (PRP) is “normalized” by the addition of platelet-poor plasma (PPP). In this study we tested the hypothesis that the large amount of PPP required to “normalize” PRP in patients with thrombocytosis may result in falsely defective platelet function. To this end, we evaluated platelet aggregation in PRP samples “normalized” with either PPP or buffer in 16 patients with high platelet counts induced by myeloproliferative disorders. Comparison with the results obtained in healthy subjects demonstrated that patients had reduced platelet responses to ADP or collagen in PRP/PPP samples, but normal responses in PRP/buffer. By contrast, the majority of patients had severely defective platelet response to epinephrine independently from the methodological approach. We suggest that the reduced in vitro platelet aggregation previously described in patients with myeloproliferative disorders and thrombocytosis partially derived from a laboratory artefact.

Assessment of Heparins, Heparinoids and Direct Thrombin Inhibitors Using a New FXa-and RVV-V-Based Assay.

UFH and direct thrombin inhibitors are usually monitored using the activated partial thromboplastin time (aPTT), which has many known limitations. These include non-linear-dose response-relationships as well as a high variability among different reagents. In addition the aPTT has a low sensitivity towards low molecular weight heparin (LMWH). Newer methods such as chromogenic substrate analysis, ecarin clotting time or the Heptest assay have provided improved sensitivity and specificity, but still have not reached broad application in routine or emergency laboratories. The performance of a new universal monitoring method (Pefakit® PiCT®, Pentapharm, Basel) in detecting anticoagulant effects of anti-Xa and / or arti-IIa agents was evaluated.Methods: Measurements were performed by addition of platelet poor plasma to a reagent containing a combination of FXa, RVV-V (FV activating snake venom) and phospholipids. After 180 s the samples were recalcified and the clotting time was recorded. Plasma samples from 30 healthy blood donors and 50 patients receiving low molecular weight heparin were analyzed. To evaluate the assay precision 30 samples were analyzed in triplicate. Plasma pools from healthy volunteers and patients under oral anticoagulant treatment were spiked with unfractionated heparin, low molecular weight heparin, argatroban and pentasaccharide.Results: PiCT® clotting times of the healthy volunteers were 20.6 ± 1.2 sec. Clotting times of the patient samples ranged 29–195 sec. Correlation of anti-Xa activity to PiCT® clotting times was good (r=0.94). The mean variation coefficient of the repeated determinations was 2.6%. Addition of LMWH, UFH or Argatroban to normal plasma showed a steep increase and almost linear dose-response of the PiCT clotting times. Addition of pentasaccharide showed a non-linear dose-response with lesser increase of clotting times at concentrations above 0.75 μg/ml plasma. The addition of LMWH or UFH to plasma of patients under warfarin therapy showed moderate additive effects when compared to normal plasma, while stronger additive effects of warfarin and heparin therapy were found for argatroban.Conclusion: The prothrombinase induced clotting test (PiCT ®) is a new clotting assay, which sensitively assesses LMWH, UFH and direct thrombin inhibitors with almost linear dose-response-relationships. The results revealed a wide measuring range which allows for assessment of prophylactic, therapeutic and supratherapeutic doses of these compounds. As a clotting test with pipetting volumes and an incubation regimen identical with the aPTT, the test can be used in routine or emergency laboratories, thus facilitating 24-hour availability of the method with short turn-around times. Allowing the assessment of heparins, heparinoids as well as direct thrombin inhibitor therapy with one method may simplify their management in clinical practice.

DDAVP's shortening of the bleeding time seems due to plasma von Willebrand factor.

The pathomechanism of desmopressin (DDAVP) dependent shortening of the acetylsalicylic acid (ASA) prolonged bleeding time (BT) is still unclear. The mechanism of action is supposed to be related to the plasma levels of von Willebrand factor (vWf), but a platelet effect is also discussed. We tried to investigate whether this effect of DDAVP might be due to either plasma vWF or platelets or both. We measured the effect of DDAVP (0.4 micrograms/ml bw in 250 ml 0.9% NaCl) on the Thrombostat 4000 (in vitro bleeding test (IVBT)) after each of 16 volunteers ingested 100 mg of ASA twice. To measure the possible effect of DDAVP on plasma and platelets separately, a mixture of 800 microliters of platelet poor plasma (PPP) or 200 microliters of isolated platelets after DDAVP, respectively, was prepared and the IVBT monitored. Two hundred microliters of PPP or platelets, respectively, served as a control. In an additional experiment, vWF concentrate (12 U/ml) dissolved in 200 microliters of PPP after ASA ingestion, as described above, was added to 800 microliters of citrated whole blood from the same volunteers. The addition of PPP after DDAVP as well as the PPP or PRP after ASA enriched with vWf led to a significant shortening of the IVBT in comparison to the control, indicating that the effect of DDAVP on the prolonged BT seems to be an isolated effect of plasma vWf. By adding platelets we were not able to show a direct effect of DDAVP on platelets.

Unusual properties of platelet shape in coronary and cerebral artery disease

Platelet shape was examined in freshly fixed whole blood, using phase contrast microscopy, from 7 patients post-myocardial infarction, 9 patients undergoing elective angiography for the investigation of angina, and 17 patients with stroke. Platelet shape was abnormal in 29/33 patients and in 20 of them the distribution of platelet shape did not resemble that produced by known platelet activators, by venipuncture techniques known to produce platelet activation, nor by shearing platelet-rich plasma. In particular, the proportion of spherical platelets is too high relative to the proportion of smooth discoid shaped platelets. Addition of platelet-poor plasma from a patient post-myocardial infarction to normal platelet-rich plasma did not reproduce the abnormality in platelet shape. In contrast, for patients post-myocardial infarction platelet shape was normal in platelet-rich plasma, but the platelets were hypersensitive to ADP-induced aggregation. Incubation of citrated whole blood at 37°C resulted in an incomplete disappearance of the spherical platelets suggesting that the abnormal distribution of platelet shape consists of both a reversible and a more persistent abnormality. The reversible portion of the platelet shape abnormality seen in patients with stroke and coronary artery disease may be related to venipuncture-induced platelet activation; however, the nature of the persistent abnormality in platelet shape is presently unknown.

Growth control of cultured rat calvarium cells by platelet‐derived growth factor

Platelet-derived growth factor (PDGF), which is released during the "clotting cascade", has a mitogenic effect on a variety of mesenchymal cells including those of the periosteum. Cell culture provides a method of studying these effects. In the present study, PDGF was studied for its ability to stimulate quiescent calvarium periosteal cells to enter G1, indicated by new protein synthesis. After addition of platelet poor plasma, these cells later entered S phase, indicated by uptake of 3H-thymidine. Three hours after PDGF stimulation, there were quantitative increases of at least 18 protein bands as compared to cells left in serum starvation medium. Two of these new protein bands (31K and 45K) required transcription. These data suggest that PDGF in extravasated blood may stimulate appositional bone formation beneath intact periosteum at the site of injury.

Platelet aggregation induced by 1-desamino-8-D-arginine vasopressin (DDAVP) in Type IIB von Willebrand's disease.

Type IIB von Willebrand's disease is a distinct form of this disorder, in which there are abnormal factor VIII/von Willebrand factor multimers in plasma (but normal multimers in platelets) and heightened interaction between the von Willebrand factor and platelets in the presence of ristocetin. We have found that infusion of desmopressin acetate (1-desamino-8-D-arginine vasopressin [DDAVP]), an agent used in the treatment of von Willebrand's disease, causes platelet aggregation and thrombocytopenia in patients with Type IIB disease. In vitro, platelets in normal plasma and those obtained from patients with Type IIB disease before DDAVP infusion aggregated upon the addition of platelet-poor plasma from Type IIB patients treated with DDAVP. Platelet aggregation was associated with adsorption of multimers of factor VIII/von Willebrand factor onto the platelets and was inhibited by EDTA. We conclude that in Type IIB von Willebrand's disease, DDAVP releases an abnormal factor with platelet-aggregating properties. DDAVP should not be used to treat patients with Type IIB disease, since the presence of platelet aggregates in the circulation may be harmful.

Increased platelet phospholipase activity in diabetic subjects.

We studied platelet phospholipase activity in 35 diabetes mellitus (DM) patients and 27 age-matched controls. In the diabetics, the mean activity was 9.73±0.54 n mol/mg prot/30 sec, significantly higher (p<0.005) than in the controls (7.80±0.43). The degree of phospholipase was not significantly different between insulin-dependent and noninsulin-dependent diabetics, and between patients treated or not with insulin. DM patients with complications manifested the highest activity (11.45±1.16,n=14, p<0.005). The addition of platelet-poor plasma (PPP) to autologous 14C-arachidonate-labeled platelet suspensions generally lowered the phospholipase level. In the presence of PPP, phospholipase values were higher in diabetics with complications (p<0.05); overall, the DM values were not significantly different from the controls. Dietary therapy decreased serum lipids but not phospholipase; the reverse applied upon insulin or glibenclamide treatment. A dose-dependent decrease of phospholipase by in vitro insulin addition was observed only in diabetics; glucose had no effect on phospholipase of control platelets. The mechanism of increased phospholipase activity observed in DM is discussed.

Spontaneous Platelet Aggregation in Arterial Insufficiency: Mechanisms and Implications

To investigate the clinical implications and mechanisms of spontaneous platelet aggregation (SPA) in man, 150 normal subjects, 22 patient controls and 130 patients with vascular insufficiency were studied. SPA was negative in normal subjects and patient controls whereas it was positive in 36 of 66 (54%) patients with transient ischemic attacks, 6 of 32 (19%) patients with stable angina, 7 of 10 (70%) patients with acute myocardial infarction and 11 of 14 (80%) patients with acute peripheral arterial insufficiency. The SPA was inhibited with aspirin in vivo, and inhibited competitively in vitro by low concentrations of aspirin, 2-chloroadenosine, prostaglandin E1 or apyrase but only by high concentrations of heparin or hirudin. Addition of platelet-poor plasma from patients with positive SPA did not cause normal platelets to aggregate. Treatment of patients who had acute peripheral arterial insufficiency with aspirin and dipyridamole prevented SPA with notable clinical improvement of the ischemic changes.