Storage Pool Deficient Platelets Research Articles

Hermansky-Pudlak Syndrome: Spectrum in Oman.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder, characterized by oculocutaneous albinism, a hemorrhagic diathesis secondary to storage pool-deficient platelets, and in some patients' pulmonary fibrosis, granulomatous colitis, and immunodeficiency. To date, 11 different types of Hermansky-Pudlak syndrome were identified. HPS type 2 is distinctively characterized by severe neutropenia and recurrent sinopulmonary infections. HPS is more common in Puerto Rico, and this is the first report deciphering the genotypic spectrum of HPS in Oman. Between 2001 and 2021, 8 Omani cases with HPS (3 HPS type 2, 1 HPS type 3, and 4 HPS type 6) had been suspected clinically and confirmed through genetic mutation analysis. Patients had mild hemorrhagic phenotype, and variable platelet aggregation defects with different platelet agonists. All patients had characteristic eye manifestations. In addition, patients with HPS type 2 had severe neutropenia. Novel mutations in AP3B1(c.205-1G>C, c.12_13delTA (p.Asn4Lysfs*6) and HPS6 (c.19_20delCT (p. Leu7Alafs*168) were not reported in population variant databases. Diagnosis of HPS had markedly improved in Oman; however, increased clinician awareness is needed. A high index of suspicion and early referral for diagnosis and initiation of proper treatment might help improve outcomes.

Secreted Dense Granule Adenine Nucleotides Promote Calcium Influx and the Maintenance of Elevated Cytosolic Calcium Levels in Stimulated Human Platelets

Evidence that secreted dense granule adenine nucleotides mediate part of the agonist-induced cytosolic calcium ([Ca2+]i) responses in human platelets was obtained from comparisons of fura-2-loaded platelets from normal subjects and from patients with a form of platelet storage pool deficiency (SPD) in which the secretory dense granules and their contents are virtually absent. SPD platelets had normal initial [Ca2+]i increases induced by thrombin and the endoperoxide analog U46619, but a significantly enhanced decay of elevated [Ca2+]i levels following the initial increases. With thrombin, this enhanced [Ca2+]i decay was associated with decreased Ca2+ influx, as measured by Mn2+ quench of fura-2 fluorescence. Addition of micromolar concentrations of ADP, alone or together with ATP, after stimulation reversed the enhanced [Ca2+]i decay and increased Mn2+ quench in SPD platelets, but had no effect on these responses in normal platelets, while addition of 100-fold higher concentrations of ATP or apyrase before stimulation increased [Ca2+]i decay and decreased Mn2+ quench in normal platelets, but had little effect in SPD platelets. ATP and alpha,beta-methylene ATP, a specific agonist for P2X1 receptors, at micromolar concentrations also increased Mn2+ quench, but to lesser extents than did ADP, in SPD platelets isolated and loaded with fura-2 in the presence of apyrase. Similar effects of ADP and excess ATP were seen in U46619-stimulated platelets, but decreased Ca2+ influx could not be measured directly in SPD platelets, presumably due to the very transient influx response seen with U46619. These results suggest that secreted dense granule ADP and ATP contribute to the maintenance of elevated [Ca2+]i levels, but not to the initial [Ca2+]i increases, in stimulated human platelets, most likely via a nucleotide-specific component of Ca2+ influx which may be mediated by interactions with both P2X1 and P2Y1 purinoceptors.

Characterization of the Hermansky-Pudlak Syndrome in the Puerto Rican population

Hermansky-Pudlak Syndrome (HPS) is an autosomal recessive disorder consisting of a triad of tyrosinase-positive albinism, an accumulation of ceroid in tissues and a bleeding tendency due to storage pool deficient platelets. HPS is frequently found in the Puerto Rican population and a village of the Swiss Alps. Dense bodies analysis is considered the best way to diagnose this condition, because the majority of the HPS patients have a disminished number of granules on platelets, if they are not completely absent. In 1996 HPS gene was identified by linkage disequilibrium mapping and positional cloning. A 16 bp duplication leads to a frameshift mutation on exon 15 in the Puerto Rican patients. Frameshift mutations have also been found in Swiss, Irish and Japanese HPS patients. We have screened a total of 63 Puerto Rican patients for the 16 bp duplication and identified 30 (47.6%) positive homozygotes, 29 (46.0%) negative homozygotes and 4 (6.3%) heterozygotes. Based on the molecular and clinical data, we selected 8 duplication-negative patients whose bleeding times were higher than 15 minutes to perform exon. screening looking for other mutations in the HPS gene. We also gathered additional clinical data available and patient family history. None of these patients had mutations in the HPS gene when they were screened by non radioactive SSCP and DNA sequencing. Electron Microscope analysis of platelets from some of the duplication-negative patients revealed a wide spectrum of dense bodies abundancies ranging between 2 to 8 dense bodies. We are currently screening these patients to find a second HPS gene in the Puerto Rican population. We acknowledge support from NIH RCMI-G12 RR 0305, NIH-RO1-AR-39892, and UPR School of Medicine.

Identification of a Novel Transcript Produced by the Gene Responsible for the Hermansky–Pudlak Syndrome in Puerto Rico

Hermansky-Pudlak Syndrome (HPS) is a rare, autosomal recessive disorder that is characterized by oculocutaneous albinism, a predisposition to mild bleeding caused by storage-pool deficient platelets, and a ceroid storage disorder. A gene responsible for HPS in Puerto Rico maps to chromosome 10q2 and isolation of the gene has been reported. We have now identified a variant HPS cDNA that contains the same 5' sequence as the published HPS gene and a unique 3' sequence. Analysis of genomic DNA suggests that the two cDNA are derived from alternative transcripts of a single gene; two polyadenylated transcripts were found in normal human melanocytes, human bone marrow cells, human melanoma cells, lymphoblastoid cell lines, and megakaryocytic leukemia cells by reverse transcriptase polymerase chain reaction and northern analysis. The splicing exhibited by this gene is identical to the splicing found to produce two alternative transcripts of the Chediak-Higashi Syndrome gene, another pigment disorder exhibiting platelet storage pool deficiency. These studies show that the HPS gene on chromosome 10 is complex and may have more than one biologically active transcript.

Synergistic effect of storage pool deficient platelets and low plasma von willebrand factor on the severity of the hemorrhagic diathesis in hermansky‐pudlak syndrome

A study of 565 Puerto Rican patients with storage pool deficient (SPD) Hermansky-Pudlak syndrome (HPS) demonstrated that most HPS patients had minor bleeding episodes while others had repeated, severe hemorrhagic episodes requiring transfusion. The severity of bleeding in these latter patients could not be attributed to their SPD alone. As swine with SPD platelets and low von Willebrand factor antigen (vWF:Ag) have more severe hemorrhages than pigs with either defect alone, 146 albino patients and 46 normally pigmented patients were examined for their level of vWF:Ag. The risk of SPD HPS patients having severe, repeated bleeding episodes increased when vWF:Ag fell below 70 U/dL. Family studies indicated that low vWF:Ag levels were more frequently associated with O blood group than from a gene suppressing production or release of vWF1. HPS patients should be tested for vWF:Ag levels.

Collagen-induced rat platelet reactivity is enhanced in whole blood in both the presence and absence of dense granule secretion

Collagen induced aggregation, ATP secretion and thromboxane (TxB 2) generation of storage pool deficient platelets were compared to normal platelets of closely related rat strains. Platelet function was monitored in citrated-platelet-rich-plasma (PRP) and citrated whole blood. Wistar (W) and fawn-hooded (FH) rat strains and their F 2 hybrids were utilized. The W strain, which is ancestral to the FH strain, is not storage pool deficient while the FH strain is. This was manifested by the total lack of collagen induced ATP secretion from platelets of the FH strain while the platelets of the W strain secreted normally. Utilizing platelets from the F 2 generation of WxFH matings, the absence of dense granule secretion (ATP) from the FH platelets, as well as other platelet defects of FH rats, were shown to be associated with homozygosity for the red-eyed dilution gene [r]. The non-secreting FH platelets were utilized to determine the effects of secreted dense granule constituents upon collagen induced aggregation and TxB 2 generation. The non-secreting storage pool deficient platelets did aggregate and did generate TxB 2 upon stimulation with collagen; however, the storage pool deficient FH platelets demonstrated less TxB 2 generation and did not aggregate as effectively as the normally secreting platelets of the W strain. When evaluating collagen induced platelet function in whole blood as compared to PRP, the storage pool deficient platelets remained less reactive than normally secreting platelets, but both platelet types demonstrated enhanced aggregation and increased TxB 2 generation in whole blood.

Paradoxical constriction to platelets by arteries from rats with pulmonary hypertension

We recently described the early appearance of pulmonary hypertension in the fawn-hooded rat (FHR), an animal with platelet storage pool disease also known to develop systemic hypertension at later ages. Since mediators released from aggregating platelets influence vascular tone, we hypothesized that platelet-mediated pulmonary vascular responses in FHR may be abnormal and potentially linked to the mechanism of pulmonary hypertension. To test this we examined reactivity of isolated pulmonary arteries (PA) and thoracic aortas (Ao) from young FHR with moderately severe pulmonary hypertension but normal systemic pressures. These vessels were compared with PA and Ao from control Sprague-Dawley rat (SDR). Aggregating platelets (1,000-40,000 platelets/mm3) from FHR caused dilation of SDR PA and Ao but constriction of FHR PA and Ao. Qualitatively similar responses were also observed with platelets isolated from SDR implying that abnormal responses were not simply due to the storage pool deficiency in FHR. Response to the platelet-derived endothelium-dependent vasodilator ADP was markedly impaired in FHR PA and mildly impaired in FHR Ao. Endothelium-dependent dilation to acetylcholine, but not to A23187, was mildly impaired in FHR PA while responses to both dilators were normal in FHR Ao. Endothelium-independent dilation to sodium nitroprusside was normal in both FHR PA and Ao. Constrictor sensitivity to serotonin, but not to the thromboxane A2 mimetic U-46619, was increased in FHR PA while responses to both constrictors were normal in FHR Ao. In summary, PAs from FHR with spontaneous pulmonary hypertension exhibit paradoxical constriction to both normal and storage pool deficient platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Compartmentation of 4,6-difluoro-5HT studied by nuclear magnetic resonance in normal and CHS bovine platelets

A platelet storage pool deficiency (SPD) is present in platelets from cattle with the Chediak-Higashi syndrome (CHS). The most plausible hypothesis for the SPD is that dense granule precursors are simply not formed in CHS megakaryocytes. There is, however, evidence that some recently acquired 5-hydroxytryptamine (5HT) is located in granules and that the granules have an acidic interior. To obtain a greater understanding of the processing of 5HT by SPD platelets normal and CHS platelets were incubated with 4,6-difluoro-5HT and studied by 19F NMR at 188 mHz. Normal platelets contained 2 compartments for 4,6-difluoro-5HT as indicated by 2 well-developed resonances for each 19F. The resonances were unequal in magnitude. The predominant resonance broadened with lower temperatures and was absent in CHS bovine platelets; it was, therefore, the dense granule compartment. There was only 1 resonance for each 19F in CHS platelets. The chemical shift was identical to the minor resonance, or non-dense granule resonance, found in normal bovine platelets but the resonance width was increased, indicating that some non-dense granule 4,6-difluoro-5HT was in a more restricted environment within CHS platelets than it was in normal platelets.

Hermansky-Pudlak syndrome (HPS): An epidemiologic study

A study of albinism in Puerto Rico identified 693 persons with albinism. Among these, the type of albinism was determined in 595, Hermansky-Pudlak syndrome (HPS) was found in 495. Approximately five of every six Puerto Rican albinos had HPS. The highest prevalence of HPS yet reported was in the northwestern quarter of the island where at least 1 in 1,800 persons had HPS, and approximately 1 in 21 were carriers. The HPS albino pigment phenotype was variable, and HPS albinos phenotypically resembled other types of oculocutaneous and ocular albinos. Ceroid storage was also variable. The consistent finding in HPS was storage pool deficient platelets. HPS is best diagnosed by lack of platelet dense bodies seen by electron microscopy. Evidence from family studies indicates that HPS is a distinct disorder due to the pleiotropic effects of a single gene mutation or a small deletion.

Specific correction of impaired acid hydrolase secretion in storage pool-deficient platelets by adenosine diphosphate.

Storage pool-deficient (SPD) platelets, which have decreased amounts of dense-granule and/or alpha-granule constituents, contain normal amounts of lysosomal acid hydrolases, but in some cases exhibit impaired secretion of these enzymes. We examined this impaired secretion response in SPD patients with varying extents of granule deficiencies, and determined the effects of added dense-granule constituents. Acid hydrolase secretion was impaired in patients with severe dense-granule deficiencies, but not in patients with lesser dense-granule deficiencies, including those with alpha-granule deficiencies as well. When dense-granule constituents (ADP, ATP, serotonin, Ca+2, pyrophosphate) were added to gel-filtered platelets, ADP, but none of the other constituents, completely corrected the impairment of thrombin and A23187-induced secretion in SPD platelets. The concentration of ADP required to normalize thrombin-induced secretion varied markedly, from 0.01 to 10 microM, among the individual patients. Fixation of platelets with formaldehyde before centrifugation did not prevent the enhancement of secretion by ADP. Excess ATP, which acts as a specific antagonist of ADP-mediated responses, completely blocked this enhancement of secretion in SPD platelets by ADP, and partially inhibited acid hydrolase secretion induced by low, but not high, concentrations of thrombin in normal platelets as well. Treatment of normal platelets with acetylsalicylic acid in vivo, but not in vitro, produced an impairment of acid hydrolase secretion similar in extent to that in SPD platelets, but which could not be completely corrected by added ADP. One possible explanation of these results is that the impairment of acid hydrolase secretion may be secondary to the dense-granule deficiency in SPD platelets, and that secreted ADP may potentiate the lysosomal secretion response in normal platelets as well.

The influence of lysophosphatidic acid on platelet protein phosphorylation.

Lysophosphatidic acid (LPA) is a lysophospholipid that is produced during thrombin stimulation of platelets, which can promote platelet aggregation. The mechanism of the effect of LPA was explored in normal platelets and in platelets from a patient with a storage pool deficiency (SPD). A comparison with other lysophospholipids showed that only LPA exerted significant effects to cause or potentiate platelet aggregation. Aspirin, an inhibitor of prostaglandin endoperoxide synthetase, had little effect on LPA-induced aggregation, but completely blocked LPA-induced serotonin secretion. LPA also promoted phosphorylation of myosin light chain (MLC), a 47 kilodalton (kDa) protein, and actin-binding protein. Aspirin significantly inhibited the phosphorylation of the 47-kDa and actin-binding proteins at 3-8 min after the addition of LPA, but had no effect on protein phosphorylation within the 1st min and had no significant effect on MLC phosphorylation. In SPD platelets, aspirin partially inhibited both aggregation and phosphorylation of the 47-kDa protein (less than 30% inhibition) and MLC (less than 40% inhibition) at time points of 1 min or less. The addition of ADP to SPD platelets enhanced the LPA response in platelets either pretreated or not pretreated with aspirin. Studies with SPD platelets indicate that thromboxane and secreted ADP contribute to, but are not necessary for, LPA-induced aggregation and phosphorylation. A23187 (a calcium ionophore) and LPA showed some selectivity to promote MLC as opposed to the 47-kDa protein phosphorylation, particularly at low concentrations of agonists and at earlier time points. The protein phosphorylation changes seen are consistent with a role for MLC phosphorylation in the granule centralization promoted with LPA.

ATP-ADP compartmentation in storage pool deficient platelets: correlation between granule-bound ADP and the bleeding time.

Platelets contain two major compartments of ATP and ADP, the cytosol and the dense granules. We separated the two compartments by controlled digitonin-induced cell lysis and measured both fractions directly in the platelets of 16 patients with storage pool deficiency. The total contents of ATP and ADP in the platelets of these patients was significantly lower than in normal controls. This was primarily caused by decreased amounts of ATP and ADP in the granule compartment, but also cytosolic ADP was low in these patients. In contrast, cytosolic ATP was not significantly decreased. The lower the amount of granule-bound ATP and ADP, the longer was the bleeding time in these patients. The best correlation was found with granule ADP. These data may indicate that ADP stored in platelet dense granules plays a role in the arrest of bleeding.

Serotonin accumulation in granules of storage pool-deficient platelets of Chediak-Higashi cattle.

Platelets from cattle with the Chediak-Higashi (CH) syndrome are virtually devoid of dense granules, serotonin (5-HT), and stored ATP and ADP. The present study determined how the handling of 5-HT in normal cattle platelets differed from that in CH cattle platelets. Normal and CH platelets accumulated 5-[14C]HT to the same extent. After normal and CH platelets were incubated with 5-HT for 12 h most 5-HT is still intact, indicating that it was protected from metabolism. Part of the newly acquired 5-HT in normal and CH platelets was in a pool that was rapidly released by 5 U/ml of thrombin, suggesting that 5-HT was, in part, within granules. Subcellular fractionation studies showed that, whereas most of the newly acquired 5-HT in normal platelets was located in the dense granule fractions, about one fourth was found in the lighter granule fraction that was enriched in alpha-granules. The dense granule fraction was virtually absent in CH platelets, and most of the granule 5-HT was associated with the lighter granule fraction. The mixed granule fraction from CH platelets accumulated 5-HT but the uptake was about 10% of that from normal platelets. Unlike normal granules the uptake of 5-HT by CH granules was only slightly inhibited by reserpine but was reversed by NH4Cl and nigericin treatment.

Thrombin and ionophore A23187-induced dense granule secretion in storage pool deficient platelets: evidence for impaired nucleotide storage as the primary dense granule defect

The secretion of the dense granule constituents ATP, ADP, calcium, pyrophosphate (PPi), and orthophosphate (Pi), and the release of magnesium induced by thrombin and the divalent cation ionophore A23187 have been quantitated directly in gel-filtered platelets from patients with storage pool deficiency (SPD). Both the contents and the maximal amounts of the dense granule constituents secretable by thrombin were decreased in all the patients studied, while the nonsecretable, retained amounts of these substances were identical in SPD and normal platelets. In response to both thrombin and A23187, the amounts of secretable ATP and ADP were strongly correlated in the platelets of individual patients; in contrast, secretable calcium showed no correlation with the nucleotides, and significant amounts of calcium were secreted in the total absence of nucleotide secretion in the platelets of several patients. The contents of magnesium were normal in all patients, and approximately 12% of platelet magnesium was liberated by thrombin in both SPD and normal platelets. A23187 induced the release of up to 70% of the magnesium content of normal platelets, but released significantly less (46%) magnesium from SPD platelets. Platelet aggregation induced by A23187 in platelet-rich plasma was also markedly decreased in SPD platelets. The correlations among secretable dense granule constituents suggest the presence in SPD platelets of abnormal dense granule structures that sequester calcium and other constituents but little or no adenine nucleotides, and are thus consistent with a hypothesis that impaired nucleotide transport and/or storage may be the primary dense granule defect in this disorder. In addition, these results demonstrate that certain responses to A23187 are impaired in SPD platelets.

Thrombin and Ionophore A23187-Induced Dense Granule Secretion in Storage Pool Deficient Platelets: Evidence for Impaired Nucleotide Storage as the Primary Dense Granule Defect

The secretion of the dense granule constituents ATP, ADP, calcium, pyrophosphate (PPi), and orthophosphate (Pi), and the release of magnesium induced by thrombin and the divalent cation ionophore A23187 have been quantitated directly in gel-filtered platelets from patients with storage pool deficiency (SPD). Both the contents and the maximal amounts of the dense granule constituents secretable by thrombin were decreased in all the patients studied, while the nonsecretable, retained amounts of these substances were identical in SPD and normal platelets. In response to both thrombin and A23187, the amounts of secretable ATP and ADP were strongly correlated in the platelets of individual patients; in contrast, secretable calcium showed no correlation with the nucleotides, and significant amounts of calcium were secreted in the total absence of nucleotide secretion in the platelets of several patients. The contents of magnesium were normal in all patients, and approximately 12% of platelet magnesium was liberated by thrombin in both SPD and normal platelets. A23187 induced the release of up to 70% of the magnesium content of normal platelets, but released significantly less (46%) magnesium from SPD platelets. Platelet aggregation induced by A23187 in platelet-rich plasma was also markedly decreased in SPD platelets. The correlations among secretable dense granule constituents suggest the presence in SPD platelets of abnormal dense granule structures that sequester calcium and other constituents but little or no adenine nucleotides, and are thus consistent with a hypothesis that impaired nucleotide transport and/or storage may be the primary dense granule defect in this disorder. In addition, these results demonstrate that certain responses to A23187 are impaired in SPD platelets.

Platelet malondialdehyde production and aggregation responses induced by arachidonate, prostaglandin-G2, collagen, and epinephrine in 12 patients with storage pool deficiency

We assessed the integrity of the prostaglandin synthetic pathway by measuring malondialdehyde (MDA) production and studied platelet aggregation responses to arachidonic acid and PGG2 in 12 patients with storage pool deficiency (SPD). Eight patients were deficient only in dense granules (delta-SPD) and four were deficient in both dense and alpha-granules (alpha delta-SPD). Production of MDA in response to arachidonic acid (AA), epinephrine, and collagen suggested that the transformation of AA to prostaglandin metabolites was normal in delta- SPD but abnormal in alpha delta-SPD and that the liberation of AA from phospholipids were abnormal in the majority of patients with SPD. Since the content of secretable adenosine diphosphate (ADP) is diminished in SPD platelets, the aggregation responses of these platelets to AA and PGG2 were studied to help answer the question whether these agents aggregate platelets directly or through release of endogenous ADP. Among patients with delta-SPD, aggregation by both AA and PGG2 was decreased in four albinos whose platelets were markedly deficient in ADP. In contrast, normal, or less strikingly abnormal, responses were observed in patients whose platelets either contained higher levels of platelet ADP or showed increased sensitivity to ADP. The more marked impaired responses to AA and PGG2 in patients with alpha delta-SPD suggest that substances derived from alpha-granules may also play a role in platelet aggregation by these agents. The aggregation responses in these patients with various types of SPD is consistent with a theory that granule-derived ADP mediates platelet aggregation by AA and PGG2.

Platelet Malondialdehyde Production and Aggregation Responses Induced by Arachidonate, Prostaglandin-G2, Collagen, and Epinephrine in 12 Patients With Storage Pool Deficiency

We assessed the integrity of the prostaglandin synthetic pathway by measuring malondialdehyde (MDA) production and studied platelet aggregation responses to arachidonic acid and PGG2 in 12 patients with storage pool deficiency (SPD). Eight patients were deficient only in dense granules (delta-SPD) and four were deficient in both dense and alpha-granules (alpha delta-SPD). Production of MDA in response to arachidonic acid (AA), epinephrine, and collagen suggested that the transformation of AA to prostaglandin metabolites was normal in delta-SPD but abnormal in alpha delta-SPD and that the liberation of AA from phospholipids were abnormal in the majority of patients with SPD. Since the content of secretable adenosine diphosphate (ADP) is diminished in SPD platelets, the aggregation responses of these platelets to AA and PGG2 were studied to help answer the question whether these agents aggregate platelets directly or through release of endogenous ADP. Among patients with delta-SPD, aggregation by both AA and PGG2 was decreased in four albinos whose platelets were markedly deficient in ADP. In contrast, normal, or less strikingly abnormal, responses were observed in patients whose platelets either contained higher levels of platelet ADP or showed increased sensitivity to ADP. The more marked impaired responses to AA and PGG2 in patients with alpha delta-SPD suggest that substances derived from alpha-granules may also play a role in platelet aggregation by these agents. The aggregation responses in these patients with various types of SPD is consistent with a theory that granule-derived ADP mediates platelet aggregation by AA and PGG2.

Platelet aggregation independent of adp release or prostaglandin synthesis in patients with Hermansky-Pudlak syndrome

Platelets from patients with the Hermansky-Pudlak (HPS) syndrome are deficient in the storage pool of adenine nucleotides and serotonin. As a result, the storage pool deficient (SPD) platelets develop only single waves of clumping when stimulated by threshold concentrations of aggregating agents which cause irreversible, biphasic aggregation of normal platelets. Yet, patients with HPS either have no bleeding problems or only mild symptoms.In the present study we have evaluated the importance of prostaglandin synthesis and secretion to the irreversible aggregation of HPS platelets. Results of the study demonstrate that aspirin-treated SPD platelets, which cannot form thromboxane or undergo the release reaction on stimulation by arachidonate, can still undergo irreversible aggregation in response to thrombin and ADP if treated first with epinephrine. A mechanism of membrane modulation mediated by a-adrenergic receptors cooperatively linked to the endoperoxide and thromboxane receptor can secure irreversible aggregation of normal or abnormal platelets despite absence of secretion and prostaglandin synthesis.

Biphasic Aggregation Responses to ADP and Epinephrine in Some Storage Pool Deficient Platelets: Relationship to the Role of Endogenous ADP in Platelet Aggregation and Secretion

Biphasic aggregation responses to ADP and epinephrine have been consistently observed in platelets of two patients with storage pool deficiency (SPD) despite marked reductions in platelet ADP contents. The nature of these aggregation responses was examined in relation to the role of secreted ADP as a mediator of secondary aggregation. Platelets from these patients secreted greater quantities of ADP after stimulation by epinephrine than did SPD platelets with comparable ADP deficiencies and absent second-phase aggregation, and were more sensitive than both other SPD platelets and normal platelets to the aggregating effects of low concentrations of ADP. Epinephrine-induced secondary aggregation was also associated with measurable, though less than normal, MDA formation in these patients' platelets, whereas no MDA formation occurred in SPD platelets with impaired epinephrine responses. CP/CPK did not inhibit epinephrine-induced responses in the SPD patients showing biphasic aggregation, whereas these responses in normal platelets were inhibited only with higher levels of CP/CPK than those required for inhibition of the responses to exogenous ADP. These findings suggest that secondary aggregation responses may be mediated by both prostaglandin endoperoxide production and secreted ADP, and are consistent with possibility that such mediation is primarily an intracellular process.

Direct measurement of platelet secretion in whole blood

Platelet secretion is usually measured using platelet-rich plasma (PRP) or washed platelets resuspended in buffered saline. We report here measurements of the release of adenosine triphosphate (ATP) from platelets in citrated whole blood in response to thrombin and collagen. The amount of ATP released correlates with the platelet content of the whole blood and is partially dependent on prostaglandin synthesis when release is induced by collagen. Further, the release of ATP does not occur in blood from a patient with storage-pool deficient platelets.