From bloodjournal.hematologylibrary.org by guest on April 24, 2012. For personal use only. 1980 56: 38-41 Sex-related differences in platelet aggregation: influence of the hematocrit JG Kelton, P Powers, J Julian, V Boland, CJ Carter, M Gent and J Hirsh Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved. From bloodjournal.hematologylibrary.org by guest on April 24, 2012. For personal use only. Sex-Related Differences in Platelet Influence By John A number to G. females from To examine from standard amount significantly of more to a size platelet T of the citrate distribution gating and reported associates BEEN found species aggregating These female platelets of aggregating matched from males, thrombogenesis obtained may conditions important than platelets from also observation that is uncertain. be influenced to platelet that could a fixed out modifying hematocrit. excluded ratio the Indirect from is directly reactivity influence calcium laboratories of citrate blood cells now been function to the results of in vitro in vivo. One platelet reacIt is whole with- to the patient’s that citrate is its plasma to the in the plasma concentra- hematocrit.5 citrate (age were 21-55, was pregnant, University Submitted Address McMaster IAN Department Medical (entre, September reprint concentration. Hamilton, /980 and Medical to Dr. (‘entre, Pathology. Ontario, 17. 1979; accepted requests University Medicine 3Z5. I., To John McMaster (‘anada. February G. Kelton, and 9:1, v:v), (3.8%; same and subjects Hamilton, by Grune & Stratton, female the in increased anemia. compared platelet females before and Correction Citrate (D) retested, I of Citrate is excluded plasma in can hematocrit - correction citrate were but kind of the testing, taken and the final or of any collected into into platelet citrate the sodium EDTA for count. The concentration of the hematocrit curve, of citrate determination the which applied for platelet and (Sigma ADP Inc. and stored .00/0 all experiments. and a plasma blood to 0.9 vol of a sample was was performed on concentration more were carried citrate the patient’s agents were Washington, adjusted to the sample I). at 4#{176}C. A single own batch and chloride of ADP Blood. (PRP) stored and collagen Vol. plasma diphosphate prepared 56. No. final was correc- acid-soluble and was aggre- The platelet-poor adenosine collagen in a Payton Ontario). plasma D.C.) in sodium acid-soluble out Scarborough, in the platelet-rich with diluted - Studies aggregating was the as follows: studies citrate D construct values. The adjust- studies (Appendix studies Associates, to (9 vol of blood by adding Associates, 20O,0OO/.ol formula: used determination the plasma of distribution standardize aggregation curve concentration 70#{176}C.Bovine to citrate aggregation aggregation The was accomplished A hematocrit Aggregation to Room was sodium the standardization platelet Hematocrit following over a range of hematocrit of blood for platelet The was 3.8% (Payton the I ). This specimen Platelet from (Appendix into EDTA. EDTA from into for from blood cells and its volume concentration citrate) collected Concentration be calculated concentration ment (PPP). Canada was determination to the result I I females medications On the morning 5 ml and contraceptives (see below). ted Ontario, 31) oral received 40 ml of blood and then mean was taking sampling. according METHODS 31-35, had hematocrit were was adjusted 20. 1980. 2N34. no subject to blood citrate (age No woman fasted, - 0006-4971/80/5601--0006$0/ 38 of and were sodium Since 33). for 2 wk prior Platelet the for we have AND 11 males subjects gometer From platelets and by differences associated possibility, mean was collected the ionized calcium concentration has been reported to affect platelet aggregation,6 it is possible that the reported differences in platelet aggregation between male and female platelets is an in vitro artifact caused by differences caused ). The hematocrit male is responsible MATERIALS Subjects that anticoagulant so that proportional this of female concentration. to collect amount according evidence indicates female of the a concomitantly hemoglobin Furthermore, by a variety tivity in vitro is the plasma standard practice in most into has the between the were 0.01 < for reactivity aggregation in vitro in males and after correction for the hematocrit. to threshold from age- groups.24 of platelet (p adjusted to by an artifact platelets using platelets concentrations was increased have on samples female in concentra- animals levels reported are more sensitive agents than platelets an unrelated variable male to low blood the phenomenon compared hematocrit aggregations on or collagen a similar Hirsh calcium ADP investigate reports to aggreRamwell collagen collagen is contributed frequently ionized performed and lower noted to either that higher concentration previously reactivity which of recent sensitive investigators The ADP Jack in vitro the blood:anticoagulant). of and reactivity is that and were citrate in reactivity states. more confirmed by a number ofother The relevance of in vitro tests tion curve. Gent, increased resultant (ADP) v:v. range the It is possible disease platelets a wide in platelet have aggregations . Michael observations with (9:1 occasion compartment. platelets diphosphate When difference J. Carter, these anticoagulant, over this standard a number that are agents animals.’ blood on differences in certain for anticoagulant sex-related differences in sensitivity agents of male and female platelets. of various human levels sex an Cedric female adenosine platelets but no significant in vitro HAVE male derived was reported reactivity of the Boland, human subjects. as Hematocrit explanation of the performed citrate mathematically there that possible healthy retested. The HERE we sodium than Veronica reported A 1 1 female then Julian, of distribution possibility. reactive and platelets. recently volume 3.8% Jim males. and were disappeared, the this 1 1 male subjects according have in a larger obtained Powers, age-matched results same Peter of investigators platelets tions. Kelton, of the Aggregation: (ADP) collagen. in aliquots at as described7 was used 1 (July), for 1980 From bloodjournal.hematologylibrary.org by guest on April 24, 2012. For personal use only. SEX DIFFERENCES All IN PLATELET aggregations 37#{176}Cusing (10,000 were I ml performed siliconized in the cuvettes, same aggregometer siliconized at rotating 0.8 bars rpm). The aggregometer recorder concentrations before the the maximum aggregating set at 0% and each of aggregating 0 and defined was 100% using 0 PRP maximum aggregation that agent. The aggregation agent percent experiment. were added collagen occurred percent as the maximum within wave to the at defined 5 mm ADP that 0 cuvette aggregation maximum primary Varying as of adding the aggregation was within 5 mm occurred 00 1.2 WI 1.4 of addition of the aggregating agent. Concentrations of ADP were selected that only produced a primary wave. For the purposes of these experiments, secondary aggregation was defined as an initial wave of 1.0 UJ#{176} and PPP, respectively, time 39 AGGREGATION aggregation followed by an increase in aggregation Lu 0. 1.6 of 30 greater than 5%. The initial dilution of aggregating for each aggregation observed, study. the technologist aggregating plasma agent. obtained agent was tested Statistical then of each person, for each Comparisons of dilution dilution of platelet-rich of aggregating preparation. the aggregation model aggregation, an increasing a sigmoidal-shaped formed so the dilution. The linear P = Estimates A using data. data were therefore to the logarithm was determined logit of the parameters P A and log,(P/Q) = B were the I ), in relationship to agent, “D,” invariably were using tion. The value “A” to We response and range of ADP aggregation prepared with ADP was from blood samples concentration was corrected trans- The percent maximum platelets prepared from to be logit.5 Q with obtained 50% aggregation that - I = using in aggregability 100 was was chosen rather about shifts in performed change (B) was the same the in dose whether the under whether the for both 65 to subjects concentrations. a When performed in which on platelets the citrate for hematocrit, no longer the differ- evident aggregation the unadjusted (Fig. (mean citrate “B” aggrega- than intercept the fitted the was lines. same - -- +. - - - -- - ----4 - Two in the the - 40 assumption required j--- S0 the in a 50% change dose and 2B). SE) of samples -0 00 for ± A P. weighted correction of the slope result (A) unit and following would were tested per females, to and the estimates Iog.D50 = t tests variances. prior D50, interferences unpaired formed males C draw sexes made of the dilution, equal wide female subjects aggregated did platelets from male z between estimates sample a the than of the squares. Comparisons hematocrit over from extent ence used. where a The that 60 < related transformation made observed demonstrated p < The linearly was then B log,D + 0 of aggregating was optimal model the 50 Fig. 1 . The standard line used to adjust the citrate concentration according to the hematocrit. The amount of 3.8% sodium citrate to be added to each 9 ml of whole blood is determined from the ordinate by noting where the hematocrit intersects the standard line. Platelets greater of the 22 individwere individual curve. response fitted (where concentration formed hematocrit best for each I OOP” “ responses for that of the curves percent least or lower quantities not every correction dose-response Logit limited platelet between examination The a higher 45 at random Analysis uals with and without single was chosen 40 HEMATOCRIT on the degree of aggregation selected Because from agent Depending 35 of trans- for both to produce ADP IaMI 100 B sexes. .-.cRESULTS Figure 1 illustrates standardize the ing to the size Figure correction concentration of its compartment 2 shows aggregation and prepared the final from the to accord- between concentrations with used of distribution. relationship ADP blood curve of citrate citrate for percent platelets concentration unad- justed *An on the better for hematocrit angular appropriate fit. in transformation tests, the male and female subjects. was also considered. However, based logit resulted in the transformation 1 2 3 4 5 ADP 1aMl Fig. 2. The relationship between the percent aggregation (ordinate) and final concentration of ADP (abscissa) in males (#{149}-#{149}) and females (o----o) when whole blood is collected in the standard fashion (A) and after correction for the hematocrit (B). Each point - mean ± SE; n 6. From bloodjournal.hematologylibrary.org by guest on April 24, 2012. For personal use only. KELTON 40 100 A The slopes for both the .-.cc’ o---o9 correction 00 ADP) for to platelets collected in the correction difference required 100 200 000 400 COLLAGEN 100 . DILUTION 500 600 from each z 0 60 C, Lu 40 I I 400 COLLAGEN DILUTION 500 600 700 (1/X) Fig. 3. The relationship between the percent aggregation (ordinate) and dilution of collagen (abscissa) in males (S-4) and females (o----o) when whole blood is collected in the standard fashion (A) and after correction for the hematocrit (B). Each point = mean ± SE; n 6. from male and dilution from extent subjects, when the for the female subjects plotted hematocrit (Fig. Table gation of human ences in as a function of citrate by Hardisty ago.9 They Analysis (post) Comparing Correction and a associates a relationship of premenopausal age manner. blood When the Concentration studies 0.01. tP 0.001. < were repeated Platelet the to exist, Aggregation interindividual potential yet Before not existed (pre) for Hematocrit ADP Pre Post 1.17(0.22) 1.19 Female -3.84(2.43) -3.12(1.25) 1.19(0.25) 1.23(0.23) 0.79 0.82 5.43 6.10(0.43) the log dilution of aggregating (0.52) 5.50 required -0.27 (0.53) 5.84(0.38) 1.60 3.31 agent to produce differfor be identified -2.63(1.53) Male to in the -3.20(1.19) tValue #{149}p< compared collected the citrate concentration according to the difference in platelet reactivity evident. in reactivity and Female was Male Female of the collagen was greater agent concentrations Post Sex tValue C-5O represents initially number between women when Collagen C-5O by diffen- Pre Response Slope be caused to the hematocrit.9 The results of our that the hematocrit has an important measured platelet reactivity in vitro, for and ADP induced aggregation. Platelet of similar after correcting the hematocrit, was no longer Male subjects.2 in aggre- was caused by ADP and range of aggregating a group standard of Citrate from male difference might observed been vitro of ADP necessary to initiate secondary aggreand the hematocrit, but did not attempt to this effect by adjusting the citrate concentra- differences of Statistical have in concentration Because of the considerable ences in platelet reactivity, was no longer evident following adjustment and Following agent human females aggregability platelets the tion according study indicate effect on the both collagen in 3B). 1 . Results following no significant of aggregating obtained a sex-related aggregation over a wide is shown in Fig. 3A. Once again, the the female subjects aggregated to a than did platelets from the male but this difference studies were repeated was subjects when aggregation. compared to platelets The possibility that males collagen platelets greater 50% from increased amount gation reverse 20 m concentration However, there Recently, platelets reported to have years I in the to produce suggested 200 fashion. hematocnit, female subjects DISCUSSION 80 I standard from male 700 B 100 platelets from for the (1/X) -‘---‘0w 4 different 0.001 for compared 20 C, C, significantly 40 Lu C, not 60 0 C, 4 were relationship and post- other (Table I ). In contrast, the dose of aggregating agent required to produce 50% total aggregation was significantly different (p < 0.01 for collagen, and p < z C, of the aggregation-dilution male and female precorrection ET AL. 50% aggregation. 5.86 (0.31) 5.16(0.33) 5.07t (0.20) -0.41 5.95 (0.45) 5.61 (0.51) 1.69 From bloodjournal.hematologylibrary.org by guest on April 24, 2012. For personal use only. SEX DIFFERENCES (beta error). IN PLATELET To limit 41 AGGREGATION this variation, the data reported were in a number analyzed following logistic transformation. While a significant difference (p < 0.01) in platelet reactivity existed between males and females when blood was collected in the standard fashion, no significant differ- to the associated ence existed the sample tion following size tested, reactivity could reduce this individuals be present difference to would The It and by the then the of that final citrate citrate increased findings 10-mI concentration in a blood standard be related increase indicate should be aggrega- (9:1, of whole ml of plasma and is 6.72 mg). The sample is given with v:v, blood to that a hematocrit and citrate). anticoagulant citrate amount which of 50% blood:sodium I ml of sodium total 1 was corrected sample fashion sample in Therefore, would (final of citrate would collected a contain citrate 4.5 concentration required for any blood by the formula: . Citrate volume = of blood (I x Hct) - 4.5 accord- The further blood the Our citrate curve. possible in vitro. APPENDIX be present correct for be readily the of more to a true of the patient’s hematocnit when performing platelet 108 of distribution adjusting could than To of reported sexis contrib- used to and can addition correction is volume states rather studies. laboratory by collecting whole citrate (9:0.9, v:v), performing microhematocnit, ing to the size reactivity that the influence taken into account The method is simple in a routine 3.8% sodium concentration be detected. that the aggregation in the the anticoagulant. citrate concentration a not a sample indicate in platelet to by differences employed blood into and 6%, platelet be required. These observations related differences uted correction for hematocrit. With a difference of 12% in platelet in of disease anemia sample amount of citrate collected into aggregation 2(1 to be added 0.9 ml 3.8% Hct) - - to each sodium citrate 9-mI whole is: 0.9 ml REFERENCES 1. Johnston ences M, in platelet 2. Johnson in human Freireich human M, Ramey Effects platelet Androgen Physiologist W, Ramwell aggregation. P, Drewinko EJ: PW: aggregation. platelet 3. Roper Ramwell 17:256, PW: Nature MD, of time, aggregation sex Sex and age differences concentration, Am D, Hester and J Clmn J, sex on the Pathol 71:263, I979 4. Nordoy influence inhibitory platelet 5. labelling A, Svensson B, Haycraft D, Hoak of age, sex, and the use of oral effects function. Mollison of endothelial Scand PL, red cells J Haematol Robinson with cells MA, radioactive and Hunter JC, Wiebe D: The contraceptives PGI2 21 :179, on the (prostacyclmn) of coagulant. Thromb Egan human Res CM: Divalent blood platelets 14:713, 1979 cation and the requirements role of for the anti- DA: Improved Lancet 7. Cazenave Packham to a collagen-coated method. J Lab Clin Med MA, surface: 82:978, Mustard Development JF: Adherence of of a quantitative 1973 8. Clagett GP, Collins GJ: Platelets: Thromboembolism clinical utility of antiplatelet drugs. Surg Gynecol Obstet I978 9. method 1:766, JP, platelets and the 147:257, on 1978 phosphorus. MC, Scrutton aggregation 1975 D, Johnston response. 6. differ- 1974 253:355, Hasler platelet mediated 1958 of cock Hardisty RM, H: Secondary Haematol 19:307, Hutton platelet 1970 RA, Montgomery aggregation: D, Rickard A quantitation 5, Trebilstudy. Br J