Platelet ultrastuctural morphology and its relevance in essential hypertension

-ional Journalof

CARDIOIOGY International

Journal

of Cardiology.

41 (1993) 13-20

morphology and its relevance in essential hypertension-f

Platelet ultrastuctural

Ira Pande*a,

V.K Bajpaib, M. Chandra”,

B.N Singh”

“Intensive Care Unit Department of Medicine, King George’s Medical College. Lucknow f U. PI, India 226 003 hElectron Microscopy Unir. Central Drug Research Insrirute. Lucknow ( U.PI. Indiu 226 003

(Received

22 October

1992: revision

accepted

6 April

1993)

Abstract A preliminary study of platelet ultrastuctural morphology by transmission electron microscopy in patients with established essential hypertension was carried out. Platelets of hypertensive patients were found to be in various stages of ‘platelet activation’. According to the degree of transformation observed. platelets were classified into six forms: discoid, pseudotubular, membranous, sacular. pseudopodical and hyaline and correlated to the severity of hypertension. The peudopodical and hyaline types platelets were particularly observed in severely hypertensive patients, and/or those with clinical evidence of target organ damage. Platelet ultrastructural changes may form the basis of a better understanding of the aetiology and pathogenesis of essential hypertension, and may serve as a marker in assessing the extent of underlying in vivo endothelial injury and tendency to thrombotic complications. Key words:

Platelet; Ultrastructure:

Essential hypertension

1. Introduction Various biochemical and functional platelet abnormalities have been reported and linked to the pathogenesis and complications of essential hypertension [l]. They include conflicting reports on platelet aggregation [2,3], platelet adhesiveness [4), release reaction as evident by plasma levels of beta thromboglobulin [5,6], platelet factor-4 [7,8] and serotonin kinetics [9,10], along with evidence

tPart of this work was presented at the Professor K.B. Kuwar oration organised by Indian Medical Association at King George’s Medical College, Lucknow and received best paper award for original research in 1990. * Corresponding author, at the following address: Ira Pande, Clinical Immunology Unit, Department of Medicine, All India Institute of Medical Sciences, New Delhi, I IO 029, India. Ol67-5273/93/$06.00 0 1993 Elsevier Scientific SSDI 0167-5273(93)001769-T

Publishers

Ireland

of enhanced norepinephrine efflux rate and increased calcium ion concentration [ 111: all pointing to an activated state of platelet in essential hypertension. However, available literature on these functional aspects of platelet is inconsistant, variable and does not delineate whether these functional alterations are causative or consequential to the hypertensive process [ 121. Though any functional derangement is likely to have a structural abnormality as its basis, surprisingly, very sketchy information [ 13,141 is available in literature on this important aspect. Therefore, a study was designed to evaluate changes in the ultrastructure of platelets in patients of essential hypertension, correlate to the severity of blood pressure and associated vascular complication and delineate cause-effect relationship of structural change to hypertension, if any. Ltd. All rights reserved.

I. Pande et al./Int. J. Cardiol. 41 (1993)

14

2. Patients and Methods A total of 10 patients with essential hypertension [ 151were studied. The observed platelet ultrastructural morphology was compared with the observations made earlier in the same laboratory on platelets from normotensive healthy controls. After informed consent, all these patients were subjected to thorough clinical examination, initial (routine) laboratory work up including urinalysis, serum chemistry (cholesterol, sugar, creatinine, sodium, potassium), electrocardiogram, X-ray chest and platelet count. Subjects with secondary hypertension or with associated medical disorders such as diabetes, toxaemia of pregnancy, liver disease and renal disease known to alter platelet function and subjects on platelet active drugs for the preceeding 4 weeks were excluded from this study. Severity of hypertension was graded according to World Health Organisation criteria [ 151. From each of the selected subjects, 9 ml of venous blood was slowly drawn using a disposable plastic syringe and 1%gauge needle. It was then gently transferred into a siliconised test-tube containing 1 ml buffered 3.8% sodium citrate as anticoagulant. The two were mixed by gentle turning to achieve a final volume by volume ratio of 9: 1. The samples were centrifuged at 2600 rev./min for 20 min at room temperature and subsequently the plasma was pipetted out without disturbing the buffy coat. Fixative containing 1% paraformaldehyde and 2% glutaraldehyde in 0.05 M caccodylate buffer was poured dropwise down the sides of the test tube, to avoid disturbing the buffy coat and left overnight at room temperature. The fixed buffy coat was removed with the help of a curved needle and washed with 0.05% M caccodylate buffer. The buffy coat was cut to size with fresh razor blades and osmicated in 1% osmium tetroxide in 0.05 M sodium caccodylate buffer for 1 h. fixation, samples were Following the dehydrated with graded concentrations of ethanol and embedded in a plastic mixture of araldite and epon [ 161. The sections were cut on LKB ultramicrotome using glass knives. The silver or gold color ultrathin sections were picked up on copper grids. The sections were stained with

13-20

uranyl acetate and lead citrate [ 171and were examined using a Phillips 410 LS Electron Microscope. The ultrastructural morphology of platelets by transmission electron microscopy was studied in each selected subject at the time of diagnosis of elevated blood pressure. Two of the authors assesed the transmission electron microscopy material in all the patients individually. The relative frequency with which a particular morphological form of platelet was observed to coexist was graded as l-4 on a scale of one hundred after scanning multiple fields. 3. Results 3.1. Distribution and clinico-biochemical profile of study group Table 1 shows the distribution of patients in the study group and Table 2 the mean age, blood pressure and some relevant biochemical parameters. The mean systolic and diastolic blood pressure of the study group was 198.0 and 110.1 mmHg, respectively, whereas of the control group was 123.0 and 80.3 mmHg. Platelet count of the two groups was similar. Three patients had clinical evidence of target organ damage in the form of retinopathy. 3.2. Platelet ultrastructural study Normotensive healthy controls

Observations made previously in the same laboTable I Distribution

of cases in study group

Group Total Group A Healthy controls Group B Hypertensive cases Subgroup I (mild HT. DBP 95-104 mmHg) Subgroup II (moderate HT. lO4- I 15 mmHg) Subgroup III (severe HT. > 115 mm Hg)

Number

I. Pande et al./lnr. J. Cardiol. 41 (1993)

13-20

Table 2 Mean age group, parameters

relevant

Parameter

Age BP DBP SBP Blood urea Serum creatinine Serum cholesterol Electrolytes Na K Ca Platelet count (in lac)

blood

pressure

Control Group (mean f SD.) 10.79

and

15

biochemical

Hypertensive Group (mean f S.D.) 58.4

f

14.52

60

f

80.3 123.0 28.0 0.3 168.7

f zt f

5.85 9.53 4.00 l 0.15 + 10.48

110.4 198.0 31.7 0.7 200.02

+ 9.61 f 20.44 f 36.37 f 1.18 f 26.19

133.5 ZIZ 3.08 3.3 l 0.40 10.0 f 0.18 2.2 f 0.52

135.2 3.6 10.1 2.3

f f f l

3.69 0.63 0.31 0.53 Fig. 2. Shows a normal discoid platelet with distinct membrane (PM) exhibiting all the internal characteristics cell (magnification: x 14 300).

ratory on the ultrastructure of platelets obtained from normotensive healthy subjects served as control. These normotensive platelets exhibited morphological characteristics similar to those described earlier by White [18]. Most of them appeared ovoid to spherical in shape but some of them displayed a few blunt projections (Fig. 1). Both dense bodies and alpha granules were randomly distributed throughout the cytoplasm. The open canalicular system appeared irregular in shape with empty lumina. Channels of the dense

Fig. I. Photomicrograph of normal discoid platelets. Note few platelets with blunt projections (magnification: x 12 400).

plasma of the

tubular system, few mitochondria and presence of glycogen singly or in clusters was observed throughout the cytoplasm. In addition, occasional lipid droplets and small vesicular dense bodies were noted (Figs. 2 and 3).

Fig. 3. Higher magnification of the same discoid platelet. Clearly visible is the intact dense tubular system (DTS). abundant granules (G), electron dense bodies (DB) and occasional mitochondria (M) identifiable by plication of their internal membrane into cristae (C) and lipid droplets (L). Glycogen (Gly) both singly and in clusters is visible in the cytoplasmic matrix. Note a ‘bull’s eye’ form of dense body (NB) (magnification: x 20 350).

16

1. Pande et aL/lnt. .I. Cardioi. 4f (1993)

13-M

Table 3 Morphological classification of hypertensive platelets. Type of platelet

Shape

Projections/ pseudopods

Microtubules

OCS

DTS

Organ&es

Discoid

Discoid

Absent

Normal. circumferential

Normal distribution

Randomly distributed

Pseudopodical

Spherical

Few, blunt

Membranous

Irregular

Numerous. blunt

Scarce circumferential Centralised

Sacuiar

Highly irregular, amoeboidal Totally deformed

Blunt and thin pseudopods

Appearance of microfilaments

Normal distribution empty lumen Number increased, lumen empty Lumina tilled f pseudomyelin formation Less pronounced

Numerous thin pseudopods only

Prominant microfilaments f

Decreased

Decreased

Pseudopodical Hyaline

Hypertensive cases

In contrast, platelets of hypertensive patients were seen to exhibit significant structural alterations. Ail morphological forms of platelet activation as shown in a recent report using physico-chemical agonist was observed [ 191. On the basis of the degree of structural transformation observed in relation to shape change, distribution of organelles, membranous system and arrangement of cytoskeleton elements the hypertensive platelets were classified into six forms (Table 3). Discoid. These platelets had morphological characteristics comparable to those seen in the healthy controls. They were encountered in varying frequency in all grades of hypertension. Pseudotubular. These platelets were spherical in shape with irregular outer plasma membrane, and few blunt projections. The cir~umferentia1 microtubular structure was scarce. The open canalicular system appeared increased. Dense tubular system and organelles (dense bodies, alpha granules) were distributed in the cytoplasmic matrix in a manner comparable to that observed in normal discoid platelet (Fig. 4). A few mitochondria with prominent cristae could be identified. Membranous. These platelets were irregular in shape with numerous projections. Microtubules

Decreased

Centralised

Less pronounced

Significantly decreased centralised Decreased

Complete absence, replaced by vacuoles and vesicles

disappeared from the circumferential position and were seen to form a concentric ring at cell-centres. This in turn resulted in organelles loosing their random distribution and were prominently seen drawn to the central portion of the cell; at places intimately apposed to the membranous system (Fig. 5). In addition pseudomyelin formation of membranous system was seen (Fig. 6). Empty

Fig.

4.

Photomicrograph of a pseudotubular (magni~~ation: x 14 000).

platelet

I. Pande et alhr.

J. Cardiol. 41 (1993)

13-20

Fig. 5. Photomicrograph of a membranous platelet (magnification: x 11 200). Note the granules (G) intimately apposed to the membranous system (OCSDTS).

Fig. 7. Photomicrograph of a sacular platelet. Note the numerous blunt and thin pseudopods (P) protruding in all directions (magnification: x I8 000).

lumina of open canalicular system were now seen filled with fibrin-like material. Saculur. These platelets were highly irregular and amoeboidal in shape and displayed both blunt and thin pseudopods protruding in all directions (Fig. 7). Both dense bodies and alpha granules were significantly decreased in number and open canalicular system and dense tubular system were

less pronounced activation.

Fig. 6. Photomicrograph shows a membranous platelet with prominent OCS. Note the presence of pseudomyelin formation ( -). (magnification: x 26 300).

as compared to earlier stages of

Pseudopodical. These platelets were totally and demonstrated numerous deformed pseudopods of varying length and shape in different directions. This increase in the number and length of pseudopods was seen accompanied by significant decrease in the sacular-canalicular sys-

Fig. 8. Photomicrograph of a pseudopodical platelet: totally deformed with numerous long pseudopods (P) and prominent microfibriles (MT) at terminal end. The sacular-canalicular system is scarce and granules are scanty and centralised (CO) (magnification: x I6 000).

I. Pande et alht.

18

J. Cardiol. Cl (19931 13-N

It was observed that in a particular grade of hypertension one of above the subtypes predominated, although other forms did coexist. On further analysis (Table 4) it was observed that pseudopodical and hyaline morphological types predominated in the three patients with severe hypertension and clinical evidence of end organ damage (malignant hypertensives) wheras pseudotubular and membranous types predominated in those with mild to moderate hypertension. 4. Discussion

Fig. 9. Photomicrograph shows a hyaline platelet, devoid of all cytoplasmic organelles. In place cytoplasmn exhibits empty vesciles and vacuoles (V) throughout the matrix (magnification: x 12 000).

tern and granule population. A prominent microfibrillar structure was seen extending into the terminal ends of pseudopods (Fig. 8). Some of the pseudopodical platelets showed complete absence of alpha granules, dense bodies and membranous system - the ‘prehyaline ‘stage of platelet activation. Hyaline. These platelets were totally devoid of all cytoplasmic organelles - both dense bodies and alpha granules and also displayed complete absence of mitochondria and membranous system. In place, cytoplasm showed empty vesicles or vacuoles throughout the matrix (Fig. 9).

Table 4 Qualitative pressure.

analysis

Morphology

Discoid Pseudotubular Membranous Sacular Psuedopodical Hyaline

of platelets

of platelet

in relation

Range

to diastolic

of diastolic

BP (mmHg)

95-104

104-115

4 3 3 I

2 2 7 2

2 2

I

2 1

3 3

f

blood

>I15 l

I

Despite its universal prevalence, easy detectability and effective controkessential hypertension even to this day has an unidentified aetiology [ 151. Platelets have been seen to share several common features with vascular smooth muscle cells [20] - the chief determinant of total peripheral resistance. Also, they have attained a nuclear position in this field of active research having been implicated in the aetiopathogenesis of essential hypertension by virtue of their functional alterations recorded by large number of workers in the past [2-111. Details of the structural alterations have however not been worked out, although it is logical to conceive these in view of the functional alterations reported. With this idea in mind the present study was carried out. The present study shows that platelets in the face of hypertension undergo significant structural alterations. The cellular features displayed by them are comparable to the stages of tranformation seen when platelets are activated by physicochemical agents in vitro. Interestingly, heterogeneity is observed in the degree of activation as evident by coexistance of different morphological types of platelets in different grades of hypertension. This may be suitably explained on the basis that the degree to which the ultrastructural changes occur in response to a particular stimulus depends upon the nature of agonist, its intensity and also the age of the platelet [ 181. On extrapolation of these observations in vivo, direct morphological evidence of the ongoing process of dynamic platelet activation occuring under the face of hypertension is evident. Another noteworthy feature is the relationship of the degree of tranformation observed to the se-

I. Pande et alAnt. J. Cardiol. 41 (1993)

19

13-20

verity of hypertension and more so to the associated end-organ damage. From this it can be conjectured that activation of platelets in essential hypertension is only an epiphenomenon - the activation being chiefly related to extracellular mechanisms. However, at present it is difficult to definitely decide whether the platelet abnormalities observed are fundamental to the mechanism of hypertension, or the result of the vascular damage they produce. If the latter, of course,activated platelets by way of release of various vasoactive amines and enhanced contractile behaviour may be contributory to the maintenance of essential hypertension and subsequent developement of thrombotic complications. The different morphological types of platelets conform well with the earlier reported biochemical and functional abnormalities [21]. Significant absence of dense bodies (store house of calcium ions, %hydroxytryptamine, adenosine diphosphate, norepinephrine) and alpha granules (beta thromboglobulin, platelet factor-4, platelet derived growth factor noted from pseudopodical stage onwards correlates well with alterations in the biogenie amines [22]. cationic kinetics [23] and elevated plasma levels of platelet specific proteins, beta thromboglobulin [24] and platelet factor-4 [7] reported by earlier workers in severe hypertensives. Observation of preponderance of normal discoid and pseudotubular platelets with intact granules in mild to moderate hypertensives also corroborates with normal functional and biochemical parameters pointed by some workers in essential hypertension [S]. The pseudopodical stage has been considered to represent the final step of morphological transformation in the in vitro activation cascade. Increase in the number and length of pseudopods accompanying this change has been shown to enhance the aggregation process. In view of this, our finding of preponderance of pseudopodical and hyaline type of platelets in patients with severe hypertension may reflect presence of platelets with increased aggregability, ultimately leading to endothelial injury. This may have prognostic implications in clinically determining the extent of in vivo micro/macro vascular injury and associated thrombotic complications. To conclude. there are several important points

brought out by this study. Firstly, significantly altered platelet morphology reflects an activated state of platelets in vivo in patients with essential hypertension. Secondly, platelet activation in EH is in all probability only an epiphenomenon - the activation being chiefly related to extracellular mechanisms. Platelet activation and further release of organelles rich in potent vasoactive amines. enhanced contractile behaviour and aggregation may simply be contributing to the maintainence of hypertension and be responsible for developement of thrombotic complications. Thirdly. the structural alterations observed conform well to the reported functional abnormalities and throw light to reasonably explain the conflicting nature of reports published in literature. Fourthly. morphological alterations chiefly pseudopodical and hyaline types may serve as prognostic markers in assessing the extent of underlying endothelial injury and tendency to thrombosis in a given patient. Finally, the morphological alterations of platelets provides a new dimension for delineating the role of platelets in the developement of the hypertensive process and its sequelae and may open in the near future new vistas for novel antihypertensive modalities. To unravel the dilemma whether these observed alterations in platelets in essential hypertension are an inherent primary structural defect or effect of the elevated blood pressure, furtherwork is being done to study platelet morphology after normalisation of blood pressure. 5. Acknowledgements The authors acknowledge the facilities provided by Professor B.N. Dhawan, Director CDRI and appreciate the assistance provided by Madhuli in the laboratory. 6. References Buhler FR, Resink TJ. Platelet abnormalities and the pathophysiology of essential hypertension [review]. Experentia 1988; 44 (2): 94-97. Affolter H, Erne P, Buchler FR et al. Increased sensitivity of blood platelets to S-hydroxytryptamine in EH. N S Arch Pharmacol 1984; 325 (4): 337-342. Coccerini S. Fiorentini P. Platelet adhesiveness and aggregation in hypertensive patients. Acta Media Scandinamica, Suppl 1971: 525: 273-275.

I. Pande et al./lnt. J. Cardiol. 41 (1993)

20 4

6

11

12

13

14

Poplawaski A, Skorulske M, Micwiarowski S. Platelet adhesions in hypertensive cardiovascular diseases. J Atherosclerosis Res 1968; 8: 721-723. Ikeda T, Nenaka Y, Goto A et al. Effects of prazosin on platelets aggregation and plasma BTG in EH Clin. Pharmacol Ther 1988; 37: 601-605. Catalino M, Russo U, Belleti S et al. Thrombosis Haemostasis 1985; 54: 300-307. Yamanishi J, Sano H, Saito K et al. Thrombosis Haemostasis 1985; 54 (2): 539. Guicheney P, Bandomer L, Assogna G et al. Study of in vivo platelet activation in uncomplicated EH. Life Sci 1986; 40: 615-621. Bhargava KP. Raina N, Misra N et al. Uptake of serotonin by platelets in patients with EH. Life Sci 1979; 25: 195-288. Feltkamp M, Meuree KA, Godehard E. Trytophan induced lowering of blood pressure and changes of serotonin uptake by platelets in EH. Klin Wochersh 1984: 62: 1115-1119. Erne P, Mittel HE, Burgisser E et al. Measurement of receptor induced changes in intracellular free calcium in human platelet. J Receptor Res 1983; 4: 587-604. Mehta J, Mehta P, Ostrowski N. Influence of propranolol and 4 hydroxypropranolol on platelet aggregation and thromboxane A2 generation. Clin Pharmacol Ther 1983; 34: 559-564. Naftilan AJ, Dzan VJ. Loscalze J. Preliminary observations on abnormalities of membrane structure and function in EH. Hypertension 1986; 8 Suppl II: II- 174-H-l 79. Mezlumian AG, Vikhert OA. Gabbason ZA et al. Func-

15 16 I7

18

19

20

21 22

23

24

13-N

tional state of blood platelets in arterial hypertension and changes in serotonin-containing cellular granules. Kardiologica 1989; 29 (9): 13-18. Gross F, Pisa Z. Strasser T et al. Management of arterial hyprtension. (WHO) Arch Int Med 1984; 144: 1045. Mollenhouver HH. Plastic embedding mixture for use in electron microscopy. Stain Technol 1964; 39: 11 l-l 14. Reynolds ES. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 1963; 17: 208-212. White JG. The secretory processes in platelets. Cell biology of the secretory processes. Basel: Karger, 1984; 546-569. Canizares C, Vivar N, Tenesaca S. Analysis of the ultrastructure of the platelet during the process of aggregation with emphasisin the cytoskeleton and membranous changes. Micro Electron Biol Cell 1988; 12: 1-16. Erne P, Resink TJ, Burguisser E et al. Platelets and hypertension. J Cardiovascul Pharmacol 1985; 7 Suppl 6: S103-S108. De Clerck F. Blood platelets in human essential hypertension. Agents Actions. 1986; 18: 563-580. Mattiason I, Mattiason B. Hood B. The cfflux rate of norepinephrine from platelets and its relation to blood pressure. Life Sci 1979; 24: 2265-2272. Chandra M, Rastogi R, Kumar A et al. Platelet ionic calcium and serum total calcium in essential hypertension. Indian J Exp Biol 1990; 28: 96-97, Petralito A. Fiore CE, Managialiro RA et al. Beta thromboglobulin levels in different stages of arterial hypertension. Thrombosis Haemostasis 1982; 48: 241.