Jon M. Gerrard

Identification of the molecular species of lysophosphatidic acid produced when platelets are stimulated by thrombin

Platelets, when stirred with 3 U thrombin/10(9) platelets, produced significant quantities of palmitoyllysophosphatidic acid (2.17 ng/10(9) platelets), stearoyllysophosphatidic acid (2.11 ng/10(9) platelets), and arachidonoyllysophosphatidic acid (1.06 ng/10(9) platelets). When platelets were pretreated with 100 microM of the phospholipase A2 inhibitor U10029A, there was a significant decrease in thrombin-stimulated production of stearoyllysophosphatidic acid (to 0.16 ng/10(9) platelets), while arachidonoyllysophosphatidic acid production was unchanged. U10029A concomitantly increased thrombin-stimulated production of stearoyl-containing phosphatidic acid species (primarily stearoylarachidonoylphosphatidic acid) from 5.99 to 9.71 ng/10(9) platelets. The results are consistent with the concept that stearoyllysophosphatidic acid production in platelets occurs via phospholipase A2 degradation of phosphatidic acid.

Effect of dietary α-linolenic acid and its ratio to linoleic acid on platelet and plasma fatty acids and thrombogenesis

The effect of dietary α-linolenic acid (18∶3n−3) and its ratio to linoleic acid (18∶2n−6) on platelet and plasma phospholipid (PL) fatty acid patterns and prostanoid production were studied in normolipidemic men. The study consisted of two 42-d phases. Each was divided into a 6-d pre-experimental period, during which a mixed fat diet was fed, and two 18-d experimental periods, during which a mixture of sunflower and olive oil [low 18∶3n−3 content, high 18∶2/18∶3 ratio (LO-HI diet)], soybean oil (intermediate 18∶3n−3 content, intermediate 18∶2/18∶3 ratio), canola oil (intermediate 18∶3n−3 content, low 18∶2/18∶3 ratio) and a mixture of sunflower, olive and flax oil [high 18∶3n−3 content, low 18∶2/18∶3 ratio (HI-LO diet)] provided 77% of the fat (26% of the energy) in the diet. The 18∶3n−3 content and the 18∶2/18∶3 ratio of the experimental diets were: 0.8%, 27.4; 6.5%, 6.9; 6.6%, 3.0; and 13.4%, 2.7, respectively. There were appreciable differences in the fatty acid composition of platelet and plasma PLs. Nevertheless, 18∶1n−9, 18∶2n−6 and 18∶3n−3 levels in PL reflected the fatty acid composition of the diets, although very little 18∶3n−3 was incorporated into PL. Both the level of 18∶3n−3 in the diet and the 18∶2/18∶3 ratio were important in influencing the levels of longer chain n−3 fatty acid, especially 20∶5n−3, in platelet and plasma PL. Production of 6-keto-PGF1α was significantly (P<0.05) higher following the HI-LO diet than the LO-HI diet although dietary fat source had no effect on bleeding time or thromboxane B2 production. The present study showed that both the level of 18∶3n−3 in the diet and its ratio to 18∶2n−6 were important in influencing long-chain n−3 fatty acid levels in platelet and plasma PL and that prostanoid production coincided with the diet-induced differences in PL fatty acid patterns.

The protein CD63 is in platelet dense granules, is deficient in a patient with Hermansky-Pudlak syndrome, and appears identical to granulophysin.

The levels and expression of the proteins CD63 and granulophysin in platelets from control and from a Hermansky-Pudlak syndrome subject (a condition characterized by dense granule and lysosomal deficiencies and the accumulation of ceroid-like material in reticuloendothelial cells) were examined. Immunofluorescence studies indicated that anti-CD63 and anti-granulophysin antibodies recognized similar numbers of granules; coapplication of antibodies did not identify more granules than the individual antibodies. Significantly fewer granules were recognized in Hermansky-Pudlak syndrome platelets than in control using either antibody. Immunoblotting studies demonstrated that anti-CD63 and anti-granulophysin antibodies apparently recognize the same protein, which was deficient in Hermansky-Pudlak platelets. Analysis by fluorescence-activated cell sorter (FACS) showed biphasic expression of CD63 and granulophysin after thrombin stimulation of control but not Hermansky-Pudlak platelets. Anti-CD63 effectively blocked detection of the protein by anti-granulophysin using immunofluorescence, ELISA, immunoblotting, and FACS analysis. Amino-terminal sequencing over the first 37 amino acids revealed that granulophysin was homologous to CD63, melanoma antigen ME491, and pltgp40. These results suggest that granulophysin and CD63 are possibly identical proteins. This is the first report of a protein present in platelet dense granules, lysosomes, and melanocytes, but deficient in a patient with Hermansky-Pudlak syndrome.

Inherited platelet-storage pool deficiency associated with a high incidence of acute myeloid leukaemia

Summary A family with an inherited bleeding disorder extending over four generations, and multiple cases of myeloblastic and myelomonoblastic leukaemia was studied. Ten members of the family had, by history, a haemorrhagic diathesis. There were three documented cases of myeloblastic leukaemia, two documented cases of myelomonoblastic leukaemia and two more cases of leukaemia by history. In four of the cases the bleeding diathesis clearly antedated the leukaemia, in two by many years. The bleeding disorder is characterized by a long bleeding time, abnormal platelet aggregation, low platelet ADP and decreased numbers of platelet dense bodies consistent with a dense granule storage pool deficiency. The number of dense granules was decreased by immunofluorescence employing quinacrine or using an antibody to the dense granule membrane protein, granulophysin, confirming an absolute decrease in dense granule numbers rather than the presence of empty granule sacs. This congenital storage pool deficiency is associated with a high incidence of acute myeloid leukaemia in this family.

In vivo measurement of thromboxane B2 and 6-keto-prostaglandin F1 alpha in humans in response to a standardized vascular injury and the influence of aspirin.

The effects of smoking, aspirin ingestion, and sex differences on bleeding times and bleeding time thromboxane B2 and 6-keto-prostaglandin (PG)F1 alpha production were examined. Nonsmoking men produced more thromboxane B2 (3.99 +/- 0.76 ng/ml) than nonsmoking women (2.13 +/- 0.24 ng/ml). Female smokers produced more thromboxane B2 (5.01 +/- 0.97 ng/ml) than nonsmoking women. Twenty-four hours after a single dose of 600 mg aspirin, in vitro production of thromboxane B2 in response to collagen fell by 95%, whereas in vivo production of thromboxane B2 and 6-keto-PGF1 alpha in bleeding time blood fell by 87% and 66%, respectively. Subjects with the lowest absolute levels of thromboxane B2 24 hours after aspirin were also those with the longest postaspirin bleeding times. Recovery of 6-keto-PGF1 alpha production was faster than recovery of thromboxane B2 production, but 6-keto-PGF1 alpha production for most subjects was still below basal 72 hours after aspirin. The influence of two different doses of long-term aspirin (80 mg every other day and 325 mg daily) on the in vivo production of thromboxane B2 and 6-keto-PGF1 alpha was studied in normals and diabetics. After 14 days of 80 mg aspirin every other day, thromboxane B2 and 6-keto-PGF1 alpha production were both substantially inhibited (93% and 78%, respectively). After 14 days of 325 mg aspirin daily, thromboxane B2 production was similarly substantially inhibited (93%), whereas 6-keto-PGF1 alpha was significantly less affected (only 45% inhibition). Study of a second group of five normal subjects confirmed that 6-keto-PGF1 alpha production was significantly inhibited 24 hours after the first dose of 325 mg aspirin but was not significantly less than basal after 14 days of 325 mg aspirin. The results suggest that 325 mg aspirin daily is more antithrombotic compared with 80 mg every other day due to the superior preservation of prostacyclin production.

Biphasic modulation of platelet phospholipase A2 activity and platelet aggregation by mepacrine (quinacrine).

The modulation of rat platelet phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) and rat platelet aggregation by mepacrine was investigated. The 2-acyl specificity of phospholipase A2 activity was confirmed by using 1-[14C]palmitoyl-2-[3H]arachidonylphosphatidylcholine as substrate. Under optimal pH, phospholipase A2 activity was not affected by aspirin but was inhibited by indomethacin. Contrary to previous reports, a biphasic modulatory role of mepacrine on phospholipase A2 activity and platelet aggregation was demonstrated. The data suggest that platelet aggregation is mediated via phospholipase A2.

The empty sack syndrome: a platelet storage pool deficiency associated with empty dense granules.

Summary. Two sisters with lifelong bleeding tendencies were examined to determine whether their condition was associated with a platelet defect. Their platelet aggregation in response to epinephrine and collagen was abnormal, and the secretion of serotonin and ATP was markedly reduced. The platelet contents of serotonin, ADP, and ATP were all diminished and the ATP:ADP ratio was increased. Direct enumeration by whole‐mount and quinacrine‐fluorescence techniques demonstrated that the platelets from both sisters had significantly fewer dense granules than controls. These characteristics are similar to an individual with Hermansky‐Pudlak syndrome and are consistent with a platelet dense granule deficiency. In contrast, immunofluorescence studies using an antibody against the dense granule membrane protein granulophysin suggested that both sisters had numbers of granules within the normal range. Evaluation by immunoblotting and ELISA indicated the presence of normal levels of granulophysin in the platelets from both sisters: FACS analysis demonstrated the surface expression of granulophysin under conditions of selective dense granule release. These results are consistent with these sisters having a form of dense granule storage pool deficiency where the granular membranes are present but the granules have reduced contents. This observation represents a novel form of storage pool disease which we have termed the empty sack syndrome.

Histamine formed in stimulated human platelets is cytoplasmic

The localization of histamine formed by human platelets in response to agonists was evaluated. 87 +/- 5% of the histamine in a suspension of platelets exposed to phorbol-12-myristate-13-acetate (PMA) was associated with the platelet pellet. Incubation of saponin-permeabilized platelets with the intracellular histamine antagonist, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine.HCl (DPPE), released 75 +/- 3.9% of the histamine into the supernatant. Under conditions where 90% of platelet serotonin was secreted into the supernatant, the majority (80%) of platelet histamine remained associated with the pellet. The results suggest that histamine synthesized in response to agonists is largely cytoplasmic.

Activation of permeabilized platelets by inositol-1, 4, 5-trisphosphate

The effect of inositol 1,4,5-trisphosphate (IP3) was studied using human platelets permeabilized with saponin and suspended in a high potassium, Ca2+-free buffer containing 40 microM EGTA and 1.2 mM magnesium. Under these conditions IP3 stimulated aggregation at a concentration of 0.5 microM with maximum aggregation at 5.0 microM. Aggregation was associated with phosphorylation of myosin light chain and a 47,000 dalton protein, and with a change in platelet shape including granule centralization and pseudopod formation similar to changes seen when cytoplasmic calcium is raised by other means. IP3 stimulated [14C]-serotonin release from platelet dense granules, [14C]-arachidonic acid release from platelet phospholipids and production of thromboxane B2. Preincubation of platelets with aspirin which blocked thromboxane formation also inhibited protein phosphorylation, serotonin secretion and partially inhibited aggregation. These results support the concept that IP3 is a major intracellular messenger in platelets and suggests that its effects are mediated both through Ca2+ flux and thromboxane formation.

Studies of the mechanism of natural killer (NK) degranulation and cytotoxicity.

Exocytosis of cytotoxic granule contents towards bound target cells is thought to be of central importance in natural killer (NK) cell‐mediated killing. Although cellular cytotoxicity involving degranulation is thought to be calcium‐dependent, the biochemical mechanisms that mediate this granule mobilization are unknown. Inositol‐1,4,5‐tris‐phosphate (IP3), which acts to elevate internal calcium levels, and 1,2‐diacylglycerol (DAG), which activates protein kinase C, are potent second messengers that have been shown to synergistically mediate secretion in other cell types. Production of these products of inositol phospholipid metabolism has previously been demonstrated in a rat NK cell line RNK upon exposure to susceptible tumor targets. We therefore Investigated the role of IP3 and DAG in NK‐mediated cytotoxicity, specifically at the level of degranulation. Pretreatment of RNK cells with neomycin, a drug that interferes with the hydrolysis of inositol phospholipids and thus inhibits the formation of second messengers, inhibited RNK cytotoxicity against a susceptible tumor target and also inhibited RNK production of DAG in response to a similar target Natural killing exhibited by normal rat nylon wool‐nonadherent spienocytes was also inhibited by neomycin. Phorbol‐12‐myristate,13‐acetate (PMA), a phorbol ester that acts like DAG to activate protein kinase C, markedly enhanced lysis of a susceptible target cell by RNK. We evaluated whether modulation of lysis by these drugs was associated with effects on RNK degranulation by assaying the release of a granule‐specific serine esterase (BLTE) in response to PMA and the calcium ionophore A23187. These agents synergized to promote the release of BLTE, and the extent of release was dependent on the concentrations of both agents. D2O and cytochalasin B, which enhance secretion in other cells, both enhanced BLTE release from RNK cells, indicating that we were detecting BLTE released via granule secretion and not due to nonspecific causes such as cell lysis. Our findings lead us to propose that NK cells form IP3 and DAG in response to susceptible target cells and that a major function of these second messengers is to mediate the exocytosis of cytotoxic granules towards the bound target cells.