Platelet adsorptive properties and platelet extracts in thromboplastin generation

(1) Simple incubation of normal platelets in saline for 10 minutes at 37 C.markedly diminishes their activity in the thromboplastin generation test. Thisloss of activity is due to the removel of a factor(s) from the platelet and ispresent in the saline extract.

(2) These "attenuated" platelets retain their effect on the recalcificationtime, prothrombin consumption, and clotting time of whole blood.

(3) Attenuated platelets, as well as platelets thought to be qualitativelydefective, regain normal activity in the TGT after incubation in normal plasmaor in plasmas from patients with qualitative platelet defects.

(4) Attenuated platelets, in contrast to qualitatively defective platelets, arerestored to normal, as measured by the TGT, after treatment with saline extracts of normal platelets. Attenuated platelets, however, do not function normally after incubation with saline extracts of qualitatively defective platelets.

(5) The possible mechanisms involved are discussed, and it is concludedthat the phenomenon involves the adsorption of plasma factor(s) by the platelet. The nature of the plasma factor(s) is not known.

Submitted on June 25, 1957 Accepted on September 15, 1957     

The Viability of Stored Human Platelets

Viability of normal human platelets preserved for short intervals at 4 C. wasstudied by in vitro labelling with Na₂Cr⁵¹O₄. As a criterion of viability twoparameters were used: (1) the survival time of the infused platelets, and (2)the maximum percentage of platelet radioactivity which could be recoveredin the circulation after infusion. From these two parameters the platelet viability index was calculated, and for stored platelets this was expressed in percent of the value obtained with freshly prepared platelets.

After 3 hours of storage at 4 C. the platelet viability index was reduced to62 per cent. With longer periods of storage the viability of the platelets fellrapidly with viability indices of 12 per cent after 24 hours and 2 per cent after48 hours. No significant difference was seen whether the platelets were storedas whole blood, as platelet-rich plasma, or as platelet concentrates suspendedin a plasma medium. When stored in saline the platelets lost their viabilitymore rapidly and the viability index was less than 5 per cent after only 24 hours.When the platelets were stored for 24 hours in a DAS-gelatin medium, theirviability fell to insignificant levels within 24 hours.

Platelets frozen in glycerol-plasma and stored for 24 hours at —75 C. showedreduction of viability to one-fifth the value obtained with fresh, unfrozen platelets. Even without storage the frozen platelets showed similar values of viability.

From these results the following conclusions may be drawn:

1. Conventional methods of storage at 4 C. result in rapid loss of plateletviability. An inverse, almost logarithmic, relationship exists between time ofstorage at 4 C. and platelet viability. The glycerol-freezing technique, althoughbetter than most available methods, induces a great loss in platelet viability.

2. At present no method can be advised by which platelets can be preservedin viable form even for relatively short periods of time. However, it is important to know that in the absence of plasma, platelet viability is lost morerapidly than in a plasma medium.

3. From the data obtained, it seems advisable for the practice of platelettransfusion to infuse platelets within less than 3 hours after collection.

4. Methods of improving the viability of stored blood platelets based upontheir metabolic needs are strongly warranted and are now under study.

Submitted on June 6, 1960 Accepted on July 13, 1960     

Isoimmunity to Blood Platelets in the Rabbit

The occurrence of platelet isoimmunity was studied in rabbits sensitized by3 to 12 transfusions of homologous blood. Platelet isoimmunity in the transfused animals was detected and measured by two different technics: (1)determination of the survival of homologous platelets labeled in vivo withP³² and infused in the sensitized recipient rabbits; and (2) study of plateletagglutinins in the serum of the multitransfused animals.

It was found that:

1. After 3 to 12 blood transfusions, platelet sensitization, as determined byreduction in survival of homologous platelets in the transfused rabbits, occurred in most animals.

2. Survival of homologous specific platelets, i.e., platelets collected fromthe sensitizing donor and infused in the respective recipient, usually had lowervalues than the survival of homologous non-specific platelets.

3. Higher degrees of depression in survival of homologous platelets werefound in the rabbits which had received larger numbers of blood transfusions.

4. Platelet isoagglutinins could be found only in three of the five rabbitswhich had been sensitized with 12 blood transfusions and the test was stronglypositive in only one rabbit.

5. Repeat studies performed 2 months after the transfusions demonstratedpersistence of platelet isosensitization, while after 15 months isosensitizationhad greatly decreased; although in one rabbit a good degree of depression inhomologous platelet survival wa still present after this time interval.

These studies mainly demonstrate the high frequency of platelet isoimmunity in multitransfused rabbits and the inadequacy of the agglutinintest in the detection of even relatively severe degrees of platelet isosensitization.

Submitted on November 30, 1962 Accepted on February 9, 1963     

The increased effectiveness of platelet concentrates prepared in acidified plasma

Platelet concentrates prepared in acidified plasma (pH 6.5-6.7) are superiorto concentrates prepared by standard methods, and are 80-90 per cent aseffective as platelet rich plasma (PRP). The use of excess citric acid to acidifyplasma promotes resuspension of the concentrate by eliminating clumping,which is a major factor in the decreased effectiveness of standard concentrates.Analysis of posttransfusion recovery and survival of platelets reveals no evidence of platelet injury in an acid medium.

Acidification of PRP inhibits the aggregation of platelets by adenosine diphosphate (ADP). The presence of endogenous ADP may be an importantfactor in clumping during standard concentrate preparation.

A method of acidification of PRP using citric acid is described which allowspreparation of an effective concentrate from fresh whole blood without subjecting the red cells to acid pH. Reconstitution of the acidified platelet poorplasma and its native red cells increases the citrate molarity by less than 6 percent and results in minimal decrease in pH of the whole blood.

Submitted on May 6, 1965 Accepted on July 25, 1965     

Sample stability for complete blood cell count using the Sysmex XN haematological analyser

Background

Sample stability is a crucial aspect for the quality of results of a haematology laboratory. This study was conducted to investigate the reliability of haematological testing using Sysmex XN in samples stored for up to 24 h at different temperatures.

Materials and methods

Haematological tests were performed on whole blood samples collected from 16 ostensibly healthy outpatients immediately after collection and 3 h, 6 h or 24 h afterwards, with triple aliquots kept at room temperature, 4 °C or 37 °C.

Results

No meaningful bias was observed after 3 h under different storage conditions, except for red blood cell distribution width (RDW) and platelet count (impedance technique, PLT-I) at 37 °C. After 6 h, meaningful bias was observed for mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV) at room temperature, red blood cell (RBC) count, mean corpuscular haemoglobin concentration (MCHC), MCH, MCV and PLT-I at 4 °C, and RBC, RDW, MCHC, MCH and PLT-I at 37 °C. After 24 h, a meaningful bias was observed for MCHC, MCV, platelet count (fluorescent technique, PLT-F) and mean platelet volume (MPV) at room temperature, MCHC, MCV, PLT-I and MPV at 4 °C, and all parameters except RBC count and MPV at 37 °C.

Discussion

Great caution should be observed when analysing results of haematological tests conducted more than 3 h after sample collection.     

Four cases of pseudothrombocytopenia due to platelet cold agglutinins

We report 4 cases of pseudothrombocytopenia due to platelet cold agglutinins. Case 1 was a 57 y.o. female whose platelet count was 97 x 10(3)/microl. Case 2 was a 37 y.o. male with a platelet count of 96 x 10(3)/microl. Case 3 was a 74 y.o. male with a platelet count of 28 x 10(3)/microl. Case 4 was a 62 y.o. female whose platelet count was 34 x 10(3)/microl. The platelet counts in these 4 cases were decreased and blood smears showed platelet clumping in blood drawn in a tube without anticoagulant just after withdrawal, as well as in blood drawn in a tube with anticoagulant. The platelets from these patients agglutinated at a temperature below 10 degrees C (case 1 and 4) and 24 degrees C (case 2). The immunoglobulin class of the platelet cold agglutinins in cases 1, 2 and 4 was IgM. Agglutinated platelets showed no activation marker, such as CD62P, CD63 or CD40L, on the surface of the platelets. The target antigen of cold agglutinins was GPIIb-IIIa in cases 1 and 2. We considered that the detection of platelet agglutination in blood without anticoagulant is important to diagnose pseudothrombocytopenia due to platelet cold agglutinins. Although this disease is considered to be very rare, we suspect that this disease may be misdiagnosed as pseudothrombocytopenia due to the presence of an anticoagulant, and overlooked.     

Susceptibility to thrombosis in normal young,aging, cortisone-treated,heparinized and x-irradiated hamsters as tested by topical application of thrombin

1. Thrombin applied topically to the everted cheek pouch of the hamsterproduced platelet and not red thrombi in exposed, uninjured blood vessels withcirculating blood. Red thrombi were produced in stagnant blood. Thrombusformation occurred in the venules for the most part and seldom in arterioles orcapillaries.

2. An in vivo test for platelet thrombus susceptibility, based on the thrombinreaction and the resistance of the hamster to thrombosis, has been described.

3. Thrombus susceptibility, measured by the thrombin test, increased withage and during cortisone treatment, and decreased after heparin injection andfollowing large doses of whole body x-irradiation.

4. The thrombin susceptibility test could be correlated with the platelet countin x-irradiated hamsters, showing a relatively critical minimum concentrationof blood platelets (100,000/cu.mm.) required for platelet thrombosis.

5. The relationship of platelet concentration to platelet thrombus formationand predisposition to hemorrhage has been discussed.

Submitted on December 6, 1954 Accepted on January 27, 1955     

Metabolism and Function of Human Platelets Washed by Albumin Density Gradient Separation

A method for washing platelets by albumin density gradient separation, originally designed for the study of platelet coagulant activities, has been modified for platelet aggregation and metabolic studies. Platelets are sedimented into a continuous density gradient of isosmolar albumin containing apyrase to protect them from clumping and physical injury and are resuspended in calcium-free Tyrode's solution. The mean recovery of platelets after two separations relative to plateletrich plasma (PRP) was 90.3%. When small amounts of plasma were added to washed platelet suspensions, aggregation and release of [¹⁴C]5-hydroxytryptamine (5HT) in response to adenosine diphosphate (ADP) or 5HT were similar to results obtained with PRP. When fibrinogen was substituted for plasma, ADP-induced aggregation occurred but was feeble. Without added plasma or fibrinogen, platelets were refractory to ADP and insensitive to the cyclic endoperoxide analogue U44619. When both ADP and U44619 were added simultaneously, in low concentrations, to washed platelets without added plasma or fibrinogen, aggregation occurred immediately. Washed platelets were not aggregated by adrenaline, which potentiated ADP-induced aggregation. Several biochemical measurements which are sensitive indicators of cellular damage were normal in washed platelets, including [¹⁴C]adenine uptake, adenylate energy charge, hypoxanthine formation and the response of adenylate cyclase to stimulation by PGE₁ or PGD₂. Platelet coagulant activities were not made available and heparin-neutralizing activity (HNA) was not spontaneously released by the washing procedure, but the washed platelets responded normally to appropriate agents by developing coagulant activities and releasing HNA. The ultrastructure of washed platelets was similar to those in control PRP. Inclusion of apyrase in the first albumin gradient had a beneficial effect on platelet morphology, aggregation and metabolism, but washing at 37deg;C compared with 25deg;C did not. Albumin density gradient separation is a useful method for isolating platelets for aggregation and metabolic studies.     

Platelet Function in a Patient with Thrombasthenia

The platelets of a patient with congenital thrombasthenia were not aggregated by ADP, thrombin, connective tissue particles, Polybrene, or phospholipase C, and did not adhere to glass as measured either on a glass slide or byretention in a glass-bead column. Clot retraction was markedly diminished.Raising the magnesium level partially corrected clot retraction but did notrestore ADP-induced clumping. The platelets were less able to promote prothrombin consumption. Fibrinogen concentration in the supernatant of frozenand thawed platelets was low, but surface fibrinogen appeared to be normal.

The thrombasthenic platelets were normal in the following respects: concentration of ATP and glyceraldehyde-3-phosphate-dehydrogenase; adhesionto connective tissue fibers; aggregation by antiplatelet serum; microelectrophoretic mobility; isoelectric point; disc shape of platelets at 37 C.; ability ofplatelets to change shape with ADP or cold; decrease in ATP concentrationand auramine staining of granules by thrombin; release of serotonin, ADP,and other materials absorbing at 260 mµ. by thrombin or connective tissueparticles; liberation of acid phosphatase during blood clotting; and plateletFactor 5 activity.

It is concluded that responses of thrombasthenic platelets to thrombin andconnective tissue particles are normal except that the liberated ADP fails tocause aggregation. The first stage of the reaction to ADP, transformation fromdisc to spiny sphere, is normal. Still to be determined at the molecular level isthe cause(s) of failure of clot retraction and ADP-induced aggregation and therelationship of these defects to the low fibrinogen concentration of plateletextracts.

Submitted on October 1, 1965 Accepted on January 16, 1966     

A Method for Evaluating the Hemostatic Effect of Various Agents in Thrombocytopenic Rats and Mice

A simple method for measuring the control of blood loss in thrombocytopenicrats and mice is described.

The administration of lyophilized platelets, brain phospholipid extractand soya bean extract failed to correct the blood loss in the thrombocytopenicanimal.

The results obtained with this method have failed to support the effectiveness of "platelet substitutes" in in vivo systems and reaffirm the requisites ofviability and recirculation for platelet transfusions.

Submitted on June 5, 1959 Accepted on August 5, 1959     

The Relation of Blood Platelet Survival and Distribution to 14C-Serotonin Distribution and Excretion

1. The distribution of ¹⁴C-5-HT following infusion is different from that ofendogenous serotonin. One hour after infusion it is, in major part, a functionof the distribution of blood platelets.

2. The spleen in rats is the site of a pool of platelets. Based on both ¹⁴Cand platelet recovery data in normal and splenectomized animals, this poolapproximates 40 per cent of the total circulating platelets.

3. Postsplenectomy thrombocytosis may relate to removal of a platelet reservoir with shift of the platelets normally contained therein to the peripheralcirculation.

4. The shape of platelet survival curves in rats is neither strictly linear norcurvilinear but normally is determined primarily by age-related processes inthe platelet. Platelet survival curves tend to become curvilinear in thrombocytopenic animals, indicating that there is probably an additional small, fixedrandom loss of platelets from the circulation.

5. The disappearance of ¹⁴C-5-HT from blood platelets approximates but isnot completely representative of the disappearance of the platelets themselves.It is probable that elution from platelets and possibly reutilization of plateletlabel occurs.

Submitted on May 27, 1966 Accepted on September 21, 1966     

Cytotoxicity of activated platelets to autologous red blood cells

Gel-filtered human platelets exerted lytic activity on autologous red blood cells (RBC) when they were coincubated at 37 degrees C with platelet-activating agents, such as thrombin, collagen, ADP, LPS or PMA in the absence of plasma. Lysis of activated platelets themselves did not occur during the incubation period examined. Morphological observations showed that RBC exposed to thrombin-activated platelets were fragmented and/or transformed into spherocytes. This haemolytic reaction by thrombin-activated platelets did not occur at 4 degrees C, or in the presence of agents which inhibited glycolysis or elevated intracellular levels of cAMP, indicating that energy-dependent and cAMP-regulated platelet metabolism was required for this reaction. When platelets and RBC were incubated in the same vessel, but were prevented from coming into direct cell to cell contact by means of a membrane barrier, their cytotoxicity was reduced but not eliminated completely. No cytotoxic activity against RBC was detected in platelet-free supernatants obtained by centrifugation after activation of platelets with thrombin. On the contrary, activated and washed platelets retained the activity. These observations suggested that the cytotoxic activity was carried by some diffusible and easily inactivated factors, which were continuously produced and liberated from activated platelets. Cyclo-oxygenase inhibitors inhibited the haemolytic activity of thrombin-activated platelets, suggesting a role for some products of platelet-cyclo-oxygenase pathway in platelet-mediated haemolysis. These results provide the first evidence for a direct role of activated platelets in mediation of RBC-damage in the absence of any plasma factors.     

Studies of the Fate of Platelets in Rats and Man

The tissue distribution of ⁵¹Cr-labeled platelets and erythrocytes was determined in normal and asplenic rats. Two hours after injection, the distributions in tissues of RBC and platelet ⁵¹Cr were not significantly differentexcept in the spleen which contained platelet ⁵¹Cr (12 per cent), significantlyin excess of red cell ⁵¹Cr (1.4 per cent). As labeled platelets were removedfrom the bloodstream, 80.2 per cent of the injected ⁵¹Cr was deposited in thespleen, liver and marrow of normal animals. During the same interval, radioactivity in all other sites examined diminished to less than 10 per cent ofinitial values. In splenectomized animals, hepatic uptake of radioactivity wastwice that observed in normal animals.

Studies in normal human subjects using external scintillation scanningsuggest that similar quantitative considerations apply to man in that, normally, one-third of total platelets are sequestered in the liver and one-thirdin the spleen while in asplenic individuals two-thirds are destroyed in the liver.

These observations suggest that the majority of platelets are cleared fromthe circulation by the reticuloendothelial system after becoming "senescent"but they do not elucidate the nature of "senescence" or the circumstancesleading to it.

Submitted on September 6, 1968 Accepted on March 27, 1969     

The Antigenic Structure of Blood Platelets. II. Histocompatibility Antigens in Rabbit Blood Platelets

The rejection time of skin homografts was measured in rabbits previouslysensitized by one intradermal injection of homologous blood platelets whichhad been carefully prepared free of leukocytes. The graft rejection time wasdetermined by gross inspection and based on the onset of definite signs ofgraft breakdown.

In the platelet sensitized rabbits, the skin homografts from the plateletdonors ("specific" grafts) were rejected significantly earlier than in nonsensitized animals in 42 per cent of the experiments. The homografts from animalsother than the platelet donors ("nonspecific" grafts) were rejected soonerthan normal in only 10 per cent of the experiments.

The results were interpreted as demonstration that blood platelets containhistocompatibility antigens.

Submitted on December 14, 1961 Accepted on January 17, 1962     

The age of red blood cells is associated with bacterial infections in critically ill trauma patients

Background.

Blood transfusion increases the risk of nosocomial infection in trauma patients. Specific patient- and transfusion-related risk factors are largely unknown. In this study, risk factors for developing a bacterial infection after transfusion of red blood cells (RBC) or platelets were determined in a cohort of transfused critically ill trauma patients.

Material and methods.

A retrospective study was conducted in a mixed medical-surgical Intensive Care Unit (ICU) of a level-1 university trauma centre, in trauma patients who received a RBC or platelet transfusion. Patients who developed a bacterial infection after transfusion were compared to transfused controls who did not develop such an infection. Multivariable logistic regression was used to determine risk factors for infection.

Results.

Of the 7,118 patients admitted to the ICU during the study period, 196 trauma patients met the inclusion criteria. An infection developed in 56 patients (29%). Infection occurred irrespective of the administration of antibiotics as part of selective digestive tract decontamination, surgery status or Injury Severity Score. Transfusion of RBC stored for more than 14 days was associated with infection in trauma patients (odds ratio 1.038, [95% CI: 1.01–1.07], p=0.036). Neither the amount of RBC nor that of platelets was associated with onset of infection.

Conclusions.

Transfusion of RBC stored for more then 14 days is a risk factor for onset of bacterial infection after trauma, irrespective of the use of prophylactic antibiotics. Transfusion of platelets was not a risk factor. These results may contribute to designing prospective studies on transfusion of fresh RBC only in trauma patients.     

Platelet Cold Agglutinins

A "cold" platelet agglutinin is describedwhich is present in plasma and serum,is greater than 100,000 in molecularweight, appears to be a protein of the immunoglobulin type, is adsorbed by platelets in the absence of bivalent cations, andreacts only at temperatures below 34°C.It agglutinates homologous and autologous platelets but appears to have noeffect on in vivo platelet function. It isseen in patients with systemic disease andcan cause spuriously low platelet counts.

Submitted on January 15, 1970 Accepted on January 27, 1970     

Platelet functional defects in thrombocytopenic purpura

A defect in a serum thromboplastic component was found in six patients withsevere thrombocytopenic purpura in addition to the platelet thromboplasticdefect.

The defective sera returned to normal spontaneously upon storage at -20 C.The addition of washed normal platelets during coagulation completely corrected the serum defect in the one instance in which it was possible to carry outthe experiment.

It was postulated that a platelet factor was primarily at fault, resulting in thelack of immediate activation of a serum thromboplastic component.

The two thromboplastic defects are cumulative and are possibly dependentupon separate platelet factors.

Submitted on December 4, 1956 Accepted on March 7, 1957     

Thrombocytopathia Due to Abnormalities in Platelet Release Reaction--Studies on Six Unrelated Patients

The prolonged bleeding time in sixunrelated patients ages 24-63, wasattributed to impaired platelet aggregation; this could be accounted for bythe decreased release of platelet adenosine diphosphate (ADP) that was obtained in all patients. As a consequence of this "platelet release abnormality," collagen-induced platelet aggregation was impaired, the secondwave of epinephrine-induced aggregation was decreased (although not invariably absent), and the normal initialwave of ADP-induced aggregation wasfollowed by rapid disaggregation at37°C. In four patients, the abnormalityin collagen-induced aggregation andADP release appeared to be differentthan the defect produced by aspirin,whereas two patients appeared tohave an "aspirinlike defect." The platelets of all patients adhered normallyto connective tissue fibers but werepoorly retained in glass bead filters.Other abnormalities included impairedkaolin-induced platelet factor 3 availability in four patients, large platelets(which aggregated normally with bovine fibrinogen) in two, and small platelets in one patient. The general termthrombocytopathia is suggested to describe abnormalities in platelet function; where a specific defect in the release reaction has been demonstrated,"platelet release abnormality" is suggested as an appropriate, specificterm.

Submitted on April 20, 1971 Revised on August 16, 1971 Accepted on August 20, 1971     

Platelet activation by sustained exposure to low-dose plasmin.

Plasmin has been reported to activate and inhibit platelet function depending on dose and exposure temperature. The present study examines the induction of fibrinogen-dependent platelet aggregation following prolonged (60 min) platelet exposure to very low doses of plasmin (0.05 CU/ml) at either 22 or 37 degrees C. Maximum aggregation [mean +/- SD, 60 +/- 19 light transmission units (LTU); n = 43] occurred following platelet exposure to plasmin at 22 degrees C, but significant platelet aggregation (28 +/- 4 LTU, n = 3) also occurred following plasmin treatment at 37 degrees C. Plasmin-induced platelet aggregates appeared microscopically larger than aggregates of adenosine diphosphate (ADP)-activated platelets, and were less reversible. Aggregated plasmin-treated platelets also expressed more procoagulant activity than platelets aggregated with ADP, as reflected by shortening of the plasma kaolin recalcification time. Aggregation of platelets exposed to very low doses of plasmin was not accompanied by dense or alpha-granule secretion, and was unaffected by ADP antagonists or aspirin. Partial inhibition of platelet aggregation, however, was achieved with metabolic inhibitors, PGE1, and inhibitors of phosphoinositide 3-kinase or protein kinase C. Although fibrinogen was required for plasmin-treated platelet aggregation, [125I]-fibrinogen binding comprised only 58 +/- 3% (n = 3) of fibrinogen binding associated with ADP aggregated platelets. This was consistent with observed decreases in reptilase-induced fibrin clot retraction. Taken together, these data suggest that sustained exposure of platelets to very low plasmin doses leads to platelet activation and thus may contribute to thrombotic complications in vivo.     

Organizing Committee:

Platelets bind annexin V when stimulated with combinations of platelet agonists such as collagen and thrombin. Previous studies have demonstrated significant heterogeneity of platelets binding annexin V. The relative role of the thrombin protease-activated receptors (PARs), PAR1 and PAR4, together with different methods of platelet preparation on annexin V binding to platelets is unclear. We therefore investigated the role of PAR1- and PAR4-activating peptides in combination with collagen-related peptide on annexin V binding. In diluted whole blood, PAR1- and PAR4-activating peptides were as effective as thrombin in inducing annexin V binding. However, in washed platelets, PAR-activating peptides were less potent than thrombin at inducing annexin V binding. This difference was more pronounced when experiments were performed at 37°C compared to room temperature. In studies of diluted whole blood, platelet rich plasma and washed platelets, platelets incubated at room temperature bound more annexin V than platelets incubated at 37°C. We also saw a significant effect of time on annexin V binding, in that more annexin V bound to platelets with longer incubation times. In conclusion, PAR1 and PAR4-activating peptides were as effective as thrombin in inducing annexin V binding in combination with collagen-related peptide in diluted whole blood and platelet rich plasma, but not in washed platelets. In addition, incubation temperature and time has a strong influence on annexin V binding to platelets. Thus variations in these conditions may explain the differences observed between previous studies.