In vitro study of platelet concentrate preparations from whole blood using N-(2-mercaptopropionyl)-glycine

The sulfhydryl group containing drug N-(2-mercaptopropionyl)-glycine (MPG) which inhibits platelet aggregation in a reversible manner permits to prepare platelet concentrates in non-siliconized glass containers at pH 7.4. Resuspension of platelets is possible immediately after centrifugation. In vitro platelets tests were carried out after washing out the MPG in MPG-free plasma. Thereafter, no inhibitory effects on platelet functions were found. Platelets concentrated in presence of MPG were significantly better with respect to yield, maintenance of discoid shape, aggregability, and hypotonic shock response compared with control platelets concentrated in absence of MPG.     

A spectrophotometric method for following initial rate kinetics of blood platelet aggregation

A double-beam recording spectrophotometer was used to assay platelet aggregation. Agonist-induced turbidity changes, at 540 nm, in dilute suspensions of platelets (1 ml, 6-8 X 10(7) platelets) were recorded differentially against a reference cuvette, also containing platelets, as a function of time. The curves obtained showed downward pen deflections (decrease of turbidity) abolished by preincubation with the aggregation inhibitor, citrate. The turbidity decrease occurred simultaneously with microscopically determined single platelet recruitment into aggregates and its initial slope (r0) was a linear function of platelet concentration upto approximately 1.5 X 10(8) per ml. At a fixed platelet concentration the r0 values of ADP-induced aggregation of calf platelet-rich plasma varied as a hyperbolic function of ADP concentration at both 32 degrees C and 37 degrees C. The kinetic data at 37 degrees C were comparable to those, in the literature, obtained by following single platelet recruitment into aggregates. The increase in turbidity due to ADP-induced shape-change (6 +/- 1%, mean +/- S.E.M., n = 7) measured in the present study was substantially greater than that (3%) measured in the aggregometer.     

Survival in vivo of platelets stored for 48 hours in the buffycoat at 4 degrees C compared to platelet rich plasma stored at 22 degrees C

High speed centrifugation allows separation of whole blood into cell free plasma, a buffy coat and leukocyte poor red cells. The buffy coat can be used for the preparation of platelet concentrates. High lactate production at 22 degrees C requires storage of the buffy coat at 4 degrees C. Survival in vivo of platelet concentrates prepared from buffy coats stored at 4 degrees C for 48 h (BC-PC) was compared with the survival in vivo of platelet concentrates from platelet rich plasma stored at 22 degrees C for 48 h (PRP-PC). Both methods were studied in the same healthy volunteers (n = 8) using 51Cr labeled autologous platelets. The mean +/- SD recovery 15 min after reinfusion of the BC-PC was 30.5% +/- 13.3% and for PRP-PC 41.4% +/- 7.9% (p less than 0.0001). The survival in vivo for BC-PC was 2.4 days +/- 0.4 days and for PRP-PC 7.0 days +/- 1.4 days (p less than 0.0001). Since the survival in vivo is significantly less for platelets derived from the buffy coat stored at 4 degrees C, we advocate storage of platelets at 22 degrees C.     

Method for collecting rat thrombocytes and determining their aggregation

Three different methods of blood collection from rats are studied and compared, namely decapitation, catheterization of the carotid artery and puncture of the heart left ventricle. The latter method is preferable in studies of platelet aggregation. The method for isolation of washed platelets from rat blood is described in detail. The platelets stored for several hours at 20 degrees C did not lose the ability for aggregating under exposure to low concentrations of ADP. The curves of aggregation of washed and plasma platelets are provided. To study the rate of aggregation, a model is offered based on the approximation of the aggregation curves by the exponential A = A0exp (-alpha t). Such an approach made it possible to treat the data objectively and to reveal the features of platelet functional activity, particularly in arterial hypertension.     

Effect of phospholipase C (Bacillus cereus) on freshly isolated and 4-day-stored human platelets.

Phospholipase C (from Bacillus cereus) was used to study fresh and stored human platelets. Provided that the enzyme was inactivated before lipid extraction, no significant degradation of phospholipid in fresh cells was noted, even when platelets were activated or induced to change shape by ADP, collagen or thrombin. With platelets isolated from concentrates stored for transfusion for 4 days at 22 degrees C, membrane phospholipids were degraded by the enzyme to an extent depending on the pH in the platelet concentrate at day 4 of storage. The extent of phospholipid hydrolysis in platelets correlated well with the extent of release of lactate dehydrogenase during storage, with both being minimal for platelets from concentrates of final pH 6.5-6.9. Under non-lytic conditions, phosphatidylcholine was the phospholipid most degraded (40%), with no significant degradation of phosphatidylserine being detected. Storage does not seem to alter the distribution of phospholipids at the external leaflet of the plasma membrane.     

Assay of embryo-derived platelet activating factor (EDPAF) by an equine platelet aggregation assay: Preliminary data concerning its presence in bovine embryo culture media

This study was designed to establish a sensitive bioassay for bovine platelet-activating factor (PAF), to determine if the bovine embryo secretes PAF in vitro and if PAF release is correlated with the embryo's potential to establish a pregnancy. Using an equine platelet aggregation assay, lipid extracted culture media from 33 Day-7 embryos (individually cultured for 18 hours in 1 ml of Ham's F10 containing 0.4% BSA at 37 degrees C in an air: CO2 mixture of 95:5 prior to their transfer to recipient heifers) and from control media (n=15, Ham's F10+0.4% BSA incubated simultaneously without embryos) were investigated. In addition, culture media from Day-6 (n=6) and Day-1 (2-cell, n=12) bovine embryos that were cultured for 4 hours but not transferred were examined. The aggregation assay proved to be sensitive to 5 pg of PAF. The assay proved to be specific, since the PAF receptor antagonist SRI 63-441 inhibited platelet aggregation induced by culture media in dosages comparable to aggregation induced by synthetic PAF18. From the 15 Day-7 embryos that established a pregnancy 2 contained measurable amounts of PAF in their culture media. No PAF was detected in the culture media from 13 embryos that succeeded, in the 18 embryos that failed to establish a pregnancy, or in the control media. One of 6 Day-6 embryos and 3 of 12 Day-1 (2-cell) embryos secreted detectable amounts of PAF into the culture media. Although the results indicate that some bovine embryos release PAF or a PAF-like substance in vitro, PAF measurements in the culture medium seem not to be a suitable method for the evaluation of bovine embryos prior to transfer.     

C1q (c1) receptor on human platelets: inhibition of collagen-induced platelet aggregation by C1q (C1) molecules.

Hemolytically active human C1q incubated with EA before the addition of complement inhibited the immune hemolysis. On the contrary, heat-inactivated preparation (30 min 56 degrees C) was ineffective. Preincubation of EA with bovine collagen also resulted in a decreased hemolysis. When aggregation was measured by a turbidimetric method in citrated human platelet-rich plasma, it was found that hemolytically active human C1q (C1) alone does not induce platelet aggregation. However, in its presence the platelets failed to aggregate or exhibited a significantly reduced aggregation response to bovine collagen. The inhibition by C1q depended on the preincubation time with platelets. Heat treatment (30 min 56 degrees C) destroyed the inhibitory action of C1q (C1). The effect of C1q proved to be highly specific because different C1q preparations at their inhibitory doses in collagen-induced platelet aggregation did not influence the response to other aggregating agents (bovine thrombin, ADP, horse anti-human thymocyte globulin, goat anti-baboon platelet antiserum). The results prove that collagen and C1q are capable of binding to the same site(s); namely, to those of EA and human platelets; furthermore, they suggest the presence of a receptor for C1q (C1) on human platelets.     

Influence of temperature on blood coagulation in vitro (author's transl)]

The influence of different temperatures between 13 degrees C and 45 degrees C on coagulation factors in vitro was studied by measuring clotting time with the recalcification time, partial thromboplastin time (PTT), and thromboplastin time test. In all three tests the shortest clotting times were measured at a temperature of 40 degrees C. The relation between temperature and clotting time was similar in fresh plasma and in plasma which had been stored at a temperature of --20 degrees C before examination. However, in all tests stored plasma showed shorter coagulation times. Prolongation of coagulation time at 45 degrees C is caused by irreversible reduction of coagulation activity in the plasma. At the same time thromboplastin- and PTT-reagent are imparied in their coagulation acitvity by a temperature of 45 decrees C. In comparison to plasma obtained from healthy persons plasma from patients with hemophilia A or B or with v. Willebrand's disease reacted more sensitive to changes in temperature in the PTT test. The coagulation defect was definitely more pronounced at 27 degrees and 17 degrees C than at 37 degrees C. It was not possible to differentiate these three coagulopathies with the PTT test at 27 degrees and 17 degrees C.     

Platelet cryopreservation. 1. In vitro aggregation studies

Platelets were harvested by a Hemonetics Model-30 discontinuous cell separator from 20 normal volunteers and were cryopreserved in the presence of 5% DMSO at a controlled rate of freezing of -1 degrees C/min and stored in liquid nitrogen for up to 3 months. A significant loss of platelets occurred at the platelet concentration step through adhesion of platelets to the bag walls. A small reduction in aggregation associated with this was also seen and may reflect some damage to the platelets during the pheresis procedure. A small, but significant loss of platelet aggregation was seen with all agents following cryopreservation. Mean percentage aggregation post-thaw for all the agents was 75.4% (range 74-78%) and platelet recovery was approximately 90%. No significant changes in aggregation or recovery were seen over the 3 months' storage period. The cryoprotectant DMSO was shown to have no deleterious effect on platelet function in vitro.     

Reversible effects of glycerol on the metabolism of platelets kept at room temperature

The effects of the addition and removal of glycerol on the metabolic activities of human platelets were studied. Platelet concentrates (PC) with 20 ml plasma were stored with 3-7% (v/w) glycerol in 150-ml polyvinylchloride plastic bags for 2 days at 22 degrees C with constant agitation. Incubation of glycerol with platelets produced a dose-dependent inhibition of oxygen consumption. The inhibitions of glucose utilization and lactate production had reached the plateau level at 3% glycerol. The rate of adenosine triphosphate (ATP) generation of control platelets was 9.8 nmol/min/10(9) platelets, in which over 90% ATP generation was derived from oxidative phosphorylation. There was a dose-dependent decrease (up to 20%) by glycerol in the rate of platelet ATP generation. Glycerol inhibited glycolysis more than oxidative phosphorylation. However, the inhibition potency diminished with increasing concentrations of glycerol. The energy metabolism of platelets after removal of 5% glycerol was examined. Deglycerolized platelets after 1 hr incubation facilitated energy metabolism more strongly than that of 24 hr incubation. The platelet aggregation response to collagen was not impaired by a cycle of the addition and removal of glycerol. The results indicate that glycerol lowered the rate of ATP generation of platelets stored at 22 degrees C. However, the removal of glycerol reversed the decreased energy metabolism.     

Cryopreservation of dog platelets with dimethyl sulfoxide: therapeutic effectiveness of cryopreserved platelets in the treatment of thrombocytopenic dogs, and the effect of platelet storage at -80 degrees C

Dog platelets were frozen with 6% dimethyl sulfoxide at 2-3 degrees C per minute in a -80 degrees C mechanical freezer. The frozen platelets were stored at -80 degrees C for as long as 39 months. After storage at -80 degrees C for less than 1 year, platelet in vitro freeze-thaw-wash recovery values were 70%, and in vivo survival values 1 to 2 hr after transfusion were 40% those of fresh platelets. After 2 years or longer storage, in vitro freeze-thaw-wash recovery values were 60%, and in vivo survival values 1 to 2 hr after transfusion were 20% those of fresh platelets. These results indicate that significant deterioration of the dog platelets occurred between the first and second year of storage at -80 degrees C. Platelets that were stored frozen at -80 degrees C for less than 1 year and washed before transfusion into lethally irradiated thrombocytopenic dogs were hemostatically effective.     

Effects of gas bubbling and other forms of convection on platelets in vitro

Citrated platelet-rich human plasma was subjected to one of three experimental treatments at 37 degrees C for 15 min: stirring, bubbling (with stirring), and gentle agitation achieved by a rocking motion. The last two were "equiconvective" as judged by equilibration rates with CO2 and O2 but presumably differed in the shear stress they imposed on the cells. Stirring platelets in normal air or 5% CO2-air caused no significant aggregation. Bubbling air through platelet-rich plasma increased its pH and marked aggregation occurred. Bubbling CO2-air caused the platelet-rich plasma pH to attain its physiological level of 7.4 with less aggregation. In both cases, subsequent ADP-induced aggregation was diminished. Rocking (without stirring) in the presence of CO2-air caused negligible aggregation in platelets and an enhanced response to ADP. Because of the marked difference between the two equiconvective treatments, bubbling and rocking, the main factor in activating the human platelets is suggested to be shear stress (potentiated by high pH), with perhaps a lesser contribution from the air-plasma interface.     

Liquid storage of flow cytometrically sorted ram spermatozoa

This study investigated the optimum short-term storage conditions for ram spermatozoa before and after flow cytometric sorting. Prior to sorting, semen from four rams (n = 3 ejaculates per ram) was diluted in either a Tris-based diluent (TRIS) or AndroHep (AH) and stored at 5, 15 or 21 degrees C for 0, 6 or 24h. Sperm characteristics were assessed during storage and after sorting, freeze-thawing and incubation (6h, 37 degrees C). Functional capacity and migration ability in artificial cervical mucus (sperm migration test (SMT)) of stored, sorted and non-sorted (control) spermatozoa were assessed after freeze-thawing. After sorting, semen from three rams (n = 3 ejaculates per ram) was diluted in four different extenders: ultra-heat-treated (UHT) long life milk, TRIS containing 10% (v/v) egg yolk (TRIS-EY), AH (pH 7.4), or TEST buffer containing 10% (v/v) egg yolk (TYB). Sorted and non-sorted (control) spermatozoa were stored at 15 degrees C for 24h or 5 degrees C for 6 days. Sperm characteristics were evaluated at 0, 6 and 24h for samples stored at 15 degrees C and daily for samples stored at 5 degrees C. The SMT was performed on sorted and non-sorted (control) spermatozoa after 6h and 3 days storage at 15 and 5 degrees C, respectively. Spermatozoa stored in TRIS were sorted more efficiently, had higher motility after sorting, freezing, thawing and incubation and had greater numbers of spermatozoa penetrating into the SMT than spermatozoa stored in AH prior to sorting. Spermatozoa stored in UHT at both temperatures had higher motility, acrosome integrity and traveled greater distances in the SMT than spermatozoa stored in all other diluents. In summary, storage in TRIS at 21 degrees C was optimal for transport of ram spermatozoa to the sorting site, and storage of spermatozoa in UHT diluent (after sorting) preserved sperm viability and migration ability best at both 15 and 5 degrees C.     

In vitro effect of trichosanic acid, a major component of Trichosanthes japonica on platelet aggregation and arachidonic acid metabolism in human platelets

The in vitro effect of trichosanic acid (TCA; C18:3, omega-5), a major component of Trichosanthes japonica, on platelet aggregation and arachidonic acid (AA) metabolism in human platelets was studied. TCA dose-dependently suppressed platelet aggregation of platelet rich plasma and washed platelets. TCA decreased collagen (50 micrograms/ml)-stimulated production of thromboxane B2 (TXB2) and 12-hydroxyhepta-decatrienoic acid (HHT) in a dose-dependent manner, while that of 12-hydroxyeicosatetraenoic acid (12-HETE) was rather enhanced. The conversion of exogenously added [14C]AA to [14C]TXB2 and [14C]HHT in washed platelets was dose-dependently reduced by the addition of TCA, while that to [14C]12-HETE was increased. Similar observations were obtained when linolenic acid (LNA; C18:3, omega-3) was used. These results suggest that TCA may decrease TXA2 formation in platelets, probably due to the inhibition of cyclooxygenase pathway, and thereby reduce platelet aggregation.     

Platelet hyperactivity and abnormal Ca(2+) homeostasis in diabetes mellitus

We sought to determine the mechanisms for hyperactivity and abnormal platelet Ca(2+) homeostasis in diabetes. The glycosylated Hb (HbA(1c)) level was used as an index of glycemic control. Human platelets were loaded with Ca- green-fura red, and cytosolic Ca(2+) ([Ca(2+)](i)) and aggregation were simultaneously measured. In the first series of experiments, the platelets from diabetic and normal subjects were compared for the ability to release Ca(2+) or to promote Ca(2+) influx. A potent and relatively specific inhibitor of Na(+)/Ca(2+) exchange, 5-(4-chlorobenzyl)-2',4'-dimethylbenzamil (CB-DMB), increased the second phase of thrombin-induced Ca(2+) response, suggesting that the Na(+)/Ca(2+) exchanger works in the forward mode to mediate Ca(2+) efflux. In contrast, in the platelets from diabetics, CB-DMB decreased the Ca(2+) response, indicating that the Na(+)/Ca(2+) exchanger works in the reverse mode to mediate Ca(2+) influx. In the second series of experiments we evaluated the direct effect of hyperglycemia on platelets in vitro. We found that thrombin- and collagen-induced increases in [Ca(2+)](i) and aggregation were not acutely affected by high glucose concentrations of 45 mM. However, when the platelet-rich plasma was incubated with a high glucose concentration at 37 degrees C for 24 h, the second phase after thrombin activation was inhibited by CB-DMB. In addition, collagen-stimulated [Ca(2+)](i) response and aggregation were also increased. Thus in diabetes the direction and activity of the Na(+)/Ca(2+) exchanger is changed, which may be one of the mechanisms for the increased platelet [Ca(2+)](i) and hyperactivity. Prolonged hyperglycemia in vitro can induce similar changes, suggesting hyperglycemia per se may be the factor responsible for the platelet hyperactivity in diabetes.     

Advances in cooled semen technology.

In the horse industry, milk or milk-based extenders are used routinely for dilution and storage of semen cooled to 4-8 degrees C. Although artificial insemination (AI) with chilled and transported semen has been in use for several years, pregnancy rates are still low and variable related to variable semen quality of stallions. Over the years, a variety of extenders have been proposed for cooling, storage and transport of stallion semen. Fractionation of milk by microfiltration, ultrafiltration, diafiltration and freeze-drying techniques has allowed preparation of purified milk fractions in order to test them on stallion sperm survival. Finally, a high protective fraction, native phosphocaseinate (NPPC), was identified. A new extender, INRA96, based on modified Hanks' salts, supplemented with NPPC was then developed for use with cooled/stored semen.Four experiments were conducted to compare INRA96 and milk-based extenders under various conditions of storage. The diluted semen was maintained under aerobic conditions when stored at 15 degrees C, and anaerobic conditions when stored at 4 degrees C. In experiment 1, split ejaculates from 13 stallions were diluted either in INRA96 extender then stored at 15 degrees C or diluted in Kenney or INRA82 extenders and then stored at 4 degrees C for 24h, until insemination. In experiment 2, semen from two stallions was extended in INRA96 then inseminated immediately or stored at 15 degrees C for 3 days until insemination. In experiment 3, semen from three stallions was diluted in INRA96 then stored at 15 or 4 degrees C for 24h until insemination, finally, in experiment 4, split ejaculates from four stallions were diluted in INRA96 or E-Z Mixin extenders then stored at 4 degrees C for 24h until insemination. Experiment 1 demonstrated that at 15 degrees C, INRA96 extender significantly improved pregnancy rate per cycle compared to Kenney or INRA82 extenders at 4 degrees C after 24h of storage (57%, n=178 versus 40%, n=171, respectively; P<0.01). Experiment 2 showed that semen stored at 15 degrees C for 3 days can achieve pregnancy at a fertility rate per cycle of 48% (n=52) compared to 68% (n=50, immediate insemination, P=0.06). Experiment 3 demonstrated that INRA96 extender can be as efficient at 15 degrees C (54%, n=37) as at 4 degrees C (54%, n=35) after 24h of storage. Finally, experiment 4 showed that INRA96 extender used at 4 degrees C (59%, n=39) seems to improve fertility per cycle compared to E-Z Mixin at 4 degrees C (49%, n=39, P=0.25), but this result has to be confirmed.These results demonstrate that semen diluted in INRA96 extender and stored at 15 degrees C can be an alternative to semen diluted in milk-based extenders and stored at 4 degrees C for "poor cooler" stallions. Furthermore, INRA96 extender can be as efficient at 15 degrees C as at 4 degrees C, for preserving sperm motility and fertility.     

Phospholipid metabolism in human platelets preserved at 22 degrees C: differential effects of storage on phospholipase A2- and C-mediated reactions

The effects of preservation at 22 degrees C on phospholipid metabolism were studied in human platelets. Stimulation of fresh platelets with thrombin caused a rapid and transient rise of 1,2-diacylglycerol (DG) which was derived from phosphatidylinositol (PI) by its strictly specific phospholipase C. Lysophosphatidylcholine (lysoPC) and lysophosphatidylethanolamine (lysoPE) were also accumulated as a result of the action of phospholipase A2. No significant changes in phospholipid metabolism were detected in platelets preserved at 22 degrees C up to 6 hr. However, platelets stored for more than 12 hr showed (1) an accumulation of both lysoPC and lysoPE before thrombin activation, (2) a subsequent decrease in the formation of lysoPC and lysoPE after thrombin activation when compared to fresh platelets, (3) a threefold lower rate of liberation of arachidonic acid than fresh platelets after activation, and (4) a lower rate and extent of aggregation than fresh platelets. Nevertheless, the amount of 1,2-DG produced during preservation up to 48 hr was similar to that observed in fresh platelets. The results indicate that the markedly enhanced activity of phospholipase A2, but not phospholipase C, that occurs during platelet storage leads to the deterioration of aggregation and arachidonic acid liberation in response to thrombin.     

Correlation between the In Vitro Functionality of Stored Platelets and the Cytosolic Esterase-Induced Fluorescence Intensity with CMFDA

It has been hypothesized that the cytosolic esterase-induced fluorescence intensity (CEIFI) from carboxy dimethyl fluorescein diacetate (CMFDA) in platelets may related to platelet functions. In the present study, we measured the change of CEIFI in platelets during storage, and examined the correlations of CEIFI with the in vitro functionality of stored platelets, including the ADP-induced aggregation activity, hypotonic shock response, expression of CD62P as well as platelet apoptosis. The CEIFI of fresh platelets, when tested at 10 μM CMFDA, the mean fluorescence intensity index (MFI) was 305.9 ± 49.9 (N = 80). After 1-day storage, it was 203.8 ± 34.4, the CEIFI of the stored platelets started to decline significantly, and reduced to 112.7 ±27.7 after 7-day storage. The change in CEIFI is highly correlated to all four functional parameters measured, with the correlation coefficients being 0.9813, 0.9848, -0.9945 and -0.9847 for the ADP-induced aggregation activity, hypotonic shock response (HSR), expression of CD62P and platelet apoptosis respectively. The above results show that the CEIFI measurement of platelets represents well the viability and functional state of in vitro stored platelets. This may be used as a convenient new method for quality evaluation for stored platelets if this result can be further validated by the following clinical trials.     

Fertilizing capacity of equine spermatozoa stored for 24 hours at 5 or 20 degrees C

A breeding trial was conducted to evaluate the effect of in vitro storage time and temperature on fertilizing capacity of equine spermatozoa. Semen obtained from one stallion and diluted with skim milk-glucose extender was used to artificially inseminate 45 estrussynchronized mares. The mares were assigned to one of three treatment groups (15 mares per group): 1) insemination with fresh semen (collected within 0.5 h of use), 2) insemination with semen stored for 24 h at 20 degrees C or 3) insemination with semen stored for 24 h at 5 degrees C. The mares were inseminated daily during estrus, from the detection of a 35-mm follicle until ovulation, with 250 x 10(6) progressively motile spermatozoa (based on initial sperm motility of fresh semen). Semen samples (n = 35) were evaluated prior to insemination for percentages of total sperm motility (TSM), progressive sperm motility (PSM) and sperm velocity (SV). Single-cycle 15-d pregnancy rates. resulting from insemination with fresh semen, from fresh semen stored for 24 h at 20 degrees C or from semen stored for 24 h at 5 degrees C were the same (11 15 ; 73%). Mean diameters (mm) of 15-d embryonic vesicles were not different (P>0.05) among these three treatment groups (21.5 +/- 2.9, 19.6 +/- 2.6 and 20.5 +/- 3.6, respectively). Ten pregnant mares were aborted on Day 15 of gestation for use in another project. The pregnancy status of the 23 remaining pregnant mares was again determined at 35 to 40 d and 55 to 60 d of gestation. No pregnancy losses occurred during this time period. Mean TSM percentages were different (P<0.05) among the three groups: the fresh semen percentage was 89 +/- 2, semen stored for 24 h at 20 degrees C was 57 +/- 11 and semen stored for 24 h at 5 degrees C was 80 +/- 6. Similar differences were found for mean PSM and SV. Semen storage at either 20 or 5 degrees C for 24 h had no apparent effect on the fertilizing capacity of the extended semen samples; however, the reduction in all motility parameters tested was more dramatic in semen stored at 20 degrees C than that stored at 5 degrees C.     

Presence of factors that activate platelet aggregation in mitral stenotic patients' plasma

BACKGROUND: Although the association between mitral stenosis (MS) and increased coagulation activity is well recognized, it is unclear whether enhanced coagulation remains localized in the left atrium or whether this represents a systemic problem. To assess systemic coagulation parameters and changes in platelet aggregation, we measured fibrinogen levels and performed in vitro platelet function tests in plasma obtained from mitral stenotic patients' and from healthy control subjects' peripheral venous blood. METHODS: Sixteen newly diagnosed patients with rheumatic MS (Group P) and 16 healthy subjects (Group N) were enrolled in the study. Platelet-equalized plasma samples were evaluated to determine in vitro platelet function, using adenosine diphosphate (ADP), collagen and epinephrine in an automated aggregometer. In vitro platelet function tests in group N were performed twice, with and without plasma obtained from group P. RESULTS: There were no significant differences between the groups with respect to demographic variables. Peripheral venous fibrinogen levels in Group P were not significantly different from those in Group N. Adenosine diphosphate, epinephrine and collagen-induced platelet aggregation ratios were significantly higher in Group P than in Group N. When plasma obtained from Group P was added to Group N subjects' platelets, ADP and collagen-induced, but not epinephrine-induced, aggregation ratios were significantly increased compared to baseline levels in Group N. CONCLUSION: Platelet aggregation is increased in patients with MS, while fibrinogen levels remain similar to controls. We conclude that mitral stenotic patients exhibit increased systemic coagulation activity and that plasma extracted from these patients may contain some transferable factors that activate platelet aggregation.