Marjorie B. Zucker

Inhibition of Adenosine Diphosphate-Induced Secondary Aggregation and Other Platelet Functions by Acetylsalicylic Acid Ingestion

Summary Secondary aggregation of platelets was produced in 8 of 10 normal subjects by a critical concentration of adenosine diphosphate (ADP) ranging from 1.0-2.0 μM. Shortening of the Russell Viper Venom clotting time and release of serotonin-14C were also observed with the critical concentration of ADP or with higher levels, but did not occur in the two subjects who showed only primary aggregation in response to ADP. Ingestion of 1.3 gm of acetylsalicylic acid in a divided dose 1 and 2 hours prior to testing abolished secondary aggregation, serotonin release, and platelet factor-3 activation induced by ADP, as well as aggregation and serotonin release induced by connective tissue particles, but did not affect primary ADP-induced aggregation.

Platelet Factor 4: Production, Structure, and Physiologic and Immunologic Action

Platelet factor 4 (PF4) is a protein found in megakaryocytes and platelet α-granules (1, 2). Immunocytochemical studies show that it is present as well in mast cell granules (3) and on the endothelium of human umbilical veins, but not arteries (4). Early work on PF4 has been reviewed elsewhere (5–7). Human platelets contain about 18 ± 4 μg of PF4/109 (8). Thrombospondin, platelet-derived growth factor, and compounds derived from platelet basic protein such as β-thromboglobulin (β-TG) are found in platelet α-granules in addition to PF4. All are secreted when platelets are appropriately stimulated; for example, since thrombin is a strong stimulus, these compounds are present in much higher concentrations in serum than in plasma (e.g., 5334 vs 1.8 ng/ml for PF4 [9]). They are also secreted after contact of platelets with collagen in damaged blood vessels, for example. Production and Structure PF4 is synthesized by megakaryocytes (10), an ability that correlates with cytoplasmic maturity (2, 11). The PF4 is first packaged into vesicles and from there it is transferred to α-granules (2). Megakaryocytes express mRNA transcripts for PF4, but not for fibrinogen or albumin, which are taken up by megakaryocytes from plasma (12). Even blood platelets, which contain little mRNA, contain mRNA for PF4, whereas none is evident in human lymphocytes, cultured fibroblasts, and four types of malignant cells (13). Human PF4 is a 7.8-kDa protein that contains 70 amino acids, with two disulfide bonds, no tryptophan or methionine, two histidines, and a single tyrosine (Fig. 1). The position of its two disulfide bridges has been inferred by homology with the related compound β-TG (14). Its isoelectric point is 7.6 (15) and its extinction coefficient (1%, 280 μm) is reported to be 2.9 (3) or 5.4 (9).

Changes in Platelet Volume Produced by Temperature, Metabolic Inhibitors, and Aggregating Agents.

Summary The volume of normal human platelets, measured with a Coulter Counter, was approximately 5.8 μ3 when the platelets were disc-shaped. Platelet volume increased gradually between 15 and 30% when platelets from ACD blood were maintained at less than 10°;C or incubated with fluoride, or iodoacetate plus cyanide. Platelets from blood anticoagulated with EDTA swelled progressively; this was partially reversed in ACD plasma. Adenosine diphosphate or thrombin in concentrations causing aggregation in platelet-rich plasma induced rapid platelet swelling. ADP-induced swelling was prevented by adenosine monophosphate, but not by arcaine or EDTA, although all three compounds prevented ADP-induced clumping. Epinephrine in a concentration capable of causing aggregation did not produce swelling. All of the conditions which caused platelet swelling were associated with change in shape of platelets from smooth discs to spiny spheres. The technique described for measuring rapid changes in platelet volume should prove useful for studying membrane physiology.

Binding of factor VIII to platelets in the presence of ristocetin.

Ristocetin agglutinates platelets in platelet‐rich plasma (PRP) containing factor VIII‐related von Willebrand factor (WF). When the clumps were separated from the supernatant and resuspended in buffer, the platelets dispersed if the buffer was free of ristocetin and the PRP had been treated with aspirin or EDTA to prevent the platelet release reaction. Binding to platelets was determined by measuring WF (ristocetin cofactor assay), VIIIR‐antigen (AG, by radioimmunoassay), and coagulant activity (C) in the initial plasma, the supernatant remaining after removing the clumps formed by ristocetin, and the eluate produced after resuspending the clumped platelets in buffer. No activity was bound to platelets in the absence of ristocetin. After agglutinating platelets in PRP with ristocetin, WF, AG and C activities in the supernatants were about 65% of the initial plasma values, and in the eluates about 9%. Formalin‐fixed washed platelets resuspended in 1 in 3 plasma, and platelets in PRP diluted 1 in 3 also agglutinated when shaken with ristocetin. There was no difference between results with fixed and unfixed platelets. In one series of experiments, the supernatants had about 60% of the initial WF and AG activities; since these activities were the same, they may measure the same substance. In a later series, WF and C were compared; in shaken samples the supernatants had 39% of the initial WF activity and 56% of the initial C activity, and in unshaken samples the supernatants had 3% WF and 29% C. The significantly lower values for C binding suggest that this activity is located on a different molecule from WF‐AG and that the molecules are more readily separated in diluted than in undiluted plasma. Activity in the eluates was inversely proportional to that in the supernatants although the initial activity was never completely recovered. WF and C binding increased with platelet number and ristocetin concentration. Platelets exposed to ADP before fixation showed less agglutination than control fixed platelets and bound less WF. Fixed platelets incubated for 15 min with 30 μg trypsin/ml and washed, or platelets from a patient with the Bernard‐Soulier syndrome failed to agglutinate and bound virtually no WF, AG or C. We conclude that WF, AG and C reversibly bind to fixed or unfixed platelets in the presence of ristocetin, that binding is similar in undiluted plasma but greater for WF and AG than C in plasma diluted 1 in 3, and that ADP and trypsin alter binding, probably by affecting a platelet receptor.

Abnormal Fibrin Ultrastructure, Polymerization, and Clot Retraction in Multiple Myeloma*

We have investigated two patients with multiple myeloma who had a prolonged thrombin clotting time and absent clot retraction but no haemorrhagic diathesis. Coagulation and platelet function studies, as well as electron microscopy of the fibrin clot, indicate abnormal fibrin polymerization; adherence of myeloma protein to very thin fibrin strands results in a rigid translucent clot which fails to retract. This abnormality is not necessarily associated with a haemorrhagic diathesis.

Reversible Decrease in Platelet Retention by Glass Bead Columns (Adhesiveness) Induced by Disturbing the Blood

Summary When human blood was drawn into a syringe containing heparin and pumped through a column of glass beads, over 90% of the platelets were retained. Platelet retention was markedly reduced if the blood was first disturbed by inversion in a tube, slow centrifugation and remixing, or rapid back-and-forth transfer between two plastic or glass syringes. Retention was restored within 1 hr after the last-named maneuver but could be reduced again by redisturbing the blood. The high platelet retention noted when blood inverted in a tube was passed through columns prepared in Bowies laboratory indicates the marked effect of the plastic tubing and beads.

MECHANISMS OF PLATELET FUNCTION AS REVEALED BY THE RETENTION OF PLATELETS IN GLASS BEAD COLUMNS

In von Willebrands disease, the only in vitro abnormality of platelet function is reduced retention of platelets in glass bead columns. Although this finding suggests that the test reflects an important function in normal hemostasis, its mechanism is poorly understood. The method developed by Bowie and colleagues7 has been used to elucidate some of the variables affecting the test. Heparinized blood is passed rapidly through the column and the percentage of platelets retained in the fourth and fifth ml is calculated. In normal subjects, more than 90% is usually retained. Disturbing the blood markedly decreases retention. Retention spontaneously returns to normal in about 45 minutes after disturbance or within ten minutes after addition of apyrase. Addition of 0.1 μM ADP decreases retention with a maximum effect at ten minutes. The lowered retention caused by disturbing blood appears to result from ADP probably sequestered in the platelet membrane. Slow centrifugation and remixing of blood also reduces retention which is restored by apyrase. Platelets in platelet‐rich plasma are not retained in the column even after incubation with apyrase or apyrase plus adenosine deaminase, or after adding ADP. Adding red cell ghosts to platelet‐rich plasma improves retention, although not as effectively as erythrocytes. Since ghosts contain virtually no ADP, it is suggested that they act by altering hemodynamics, either causing platelets to enter the marginal zone of plasma so that they contact the beads and tubing, or increasing the turnover of plasma in this zone to permit greater interaction of plasma components with the foreign surfaces.

Identification and Quantitative Determination of Serotonin (5-hydroxy-tryptamine) in Blood Platelets.

Summary Serotonin (5-hydroxytryptamine) was identified in bovine platelets by the coincidence of its ultraviolet absorption spectrum with that of synthetic serotonin, and by the agreement of analyses based on ultraviolet absorption, Folin-Ciocalteu reaction and pharmacological assay using the rabbit ear and rat uterus. An average of 2.3 μg/mg of dry platelets was found, equivalent to at least 1.7 × 10−9 μg/platelet. This is more than sufficient to account for all of the serotonin found in bovine serum. No other indolic or phenolic substances were found in the platelet extracts.

Decreased Association of 45Calcium with Platelets Unable to Aggregate due to Thrombasthenia or Prolonged Calcium Deprivation

45Calcium uptake was studied with aspirin‐treated platelets that were gel‐filtered through a column of Sepharose 2B equilibrated with divalent cation‐free modified Tyrodes solution to remove readily exchangeable surface‐associated calcium. These platelets aggregated almost immediately when exposed to ADP, fibrinogen and at least 30 μm CaCl2. At this calcium ion concentration, 108 platelets took up 36.6 ± SEM 2.7 pmol of 45calcium within 1–2 min. The presence of ADP and fibrinogen did not affect the amount of calcium bound. Over 90% of this platelet‐associated calcium was removed by EDTA in 5 min suggesting that it was surface‐bound. Calcium uptake increased rapidly for 10 min, then more slowly for up to 2 h. At 60 min, maximal uptake was approached at CaCl2 concentrations between 250 and 300 μm when an average of 276 ± SEM 18 pmol of calcium was associated with 108 platelets. Only 50–60% of this calcium could be removed by EDTA in 5 min, and about 70% in 20 min, suggesting that some of it had been internalized. Platelets from two patients with thrombasthenia that were unable to aggregate took up 50% less calcium than platelets from normal volunteers. Similarly, platelets that had been incubated with EDTA at 37°C, pH 7.8 for 8 min lost the ability to aggregate despite recalcification and took up 40–60% less calcium than CaEDTA‐treated controls. Platelets from a patient with the Bernard‐Soulier syndrome aggregated and bound calcium normally. Thus the platelets’ ability to take up calcium after removal of surface‐associated calcium correlates with their ability to aggregate. Since thrombasthenic platelets and platelets rendered incapable of aggregating after prolonged calcium deprivation with EDTA do not bind fibrinogen, we postulate that some of the surface‐associated calcium normally binds to the fibrinogen receptors.

Procoagulant Activity of Platelets in Recalcified Plasma

High‐spun platelet‐poor plasma (HSPPP) and platelet‐rich plasma (PRP), prepared with minimal contact activation and pH shifts, were recalcified with little dilution in the presence of phospholipid prepared from chloroform‐extracted, acetone‐dried brain tissue. The clotting times of HSPPP and PRP were the same whether or not the platelets in PRP were first incubated or aggregated with ADP or with collagen. This suggests that platelet procoagulant activity in normal recalcified plasma derives only from provision of platelet phospholipid.