Structure and function of platelets

This chapter is relevant to Section Q1(iv) of the 2017 CICM Primary Syllabus, which expects the exam candidates to be able to "describe the process and regulation of haemostasis, coagulation and fibrinolysis." Specifically, much CICM examiner's interest in haematology seems to revolve around platelets and the drugs that inhibit their functions. Platelet-focused questions in the CICM First Part exam have consisted of the following SAQs:

• Question 3 from the first paper of 2021
• Question 6 from the first paper of 2016 
• Question 20 from the second paper of 2011
• Question 4(p.2) from the first paper of 2009

But then of course there's all the questions asking you to compare aspirin and clopidogrel. 

Linden (2016), Jurk & Kehrel (2005) or Gremmel & Frelinger III (2016) make good recommendations if anybody wants to read a more professional discussion of this topic. For most people, just Linden will be enough for basically everything except the role of platelets in haemostasis, and the haemostasis chapter probably covers that well enough.

Formation of platelets

Only mammals have platelets. It is a relatively recent invention, even though the idea of having some sort of dedicated cells to clog the woundhole appears to be a highly conserved strategy from an evolutionary standpoint. The wounded larvae of Manduca sexta and the injured sipunculid Themiste petricola all have rapid cell-mediated primary haemostasis mechanisms. Like the other lineages of blood cells, they originate from the bone marrow. The growth signals which seem to promote their differentiation from the stem cells are the granulocyte colony-stimulating factor and thrombopoietin, one of the endocrine products of the liver and kidney. The process is actually rather involved, and if the reader wishes to delve deeper,  Kaushanksy (2015) is great if you have access to Seminars in Haematology, except who does, in this economy. That's fine: you can get Schulze & Shivdasani (2005) or Change et al (2007) for free.

To summarise:

• Pluripotent haematopoietic stem cells undergo differentiation into common myeloid precursors, which could turn into megakaryocytes, red cells, mast cells or any of the monocytes.
• Megakaryocyte/erythroid progenitor cells undergo megakaryocyte lineage commitment when they decide not to become red blood cells. At this early stage they all look the same and the only way to tell them apart from the other committed progenitors is the surface expression of  β3 integrin CD61 and increased CD41 levels.  After this decision, they begin to accumulate cellular and nuclear mass, and increase in size rather impressively. Promegakaryoblasts, the first megakaryocytes which can be defined morphologically, are usually the largest cells in the marrow, with large multilobulated nuclei and a lot of granular cytoplasm. Here's a nice juicy one from the hematology.org image bank:
  
   
• Then, endomitosis take place, which is what you call this weird half-mitosis that never ends up completing the final steps of replication.  Megakaryocytes replicate their DNA but stop the process in mid-anaphase, before nuclear or cellular division (i.e. only the chromosomes and mitochondria are replicated). They do this again and again until the cell is massively polyploid (i.e. there's a gazillion chromosomes; Geddis & Kaushansky (2006) counted up to 256 sets).
• Protoplatelet formation begins in terminally mature megakaryocytes. Huge bizarre protoplasmic extensions form, full of normal-looking platelet content - including "mitochondria, ribosomes, short lengths of rough endoplasmic reticulum, secretory granules, surface connected canalicular system, and dense tubular systems" (Radley et al, 1980). Along these long processes, the occasional narrowing can be seen, where the cytoplasm is being pinched off, giving them a beaded appearance. Here's a stained microphotograph and an SEM image demonstrating the shape and scale of these things, from the same paper:
  
  These protoplasmic pseudopods extend quite far, as you can see. In fact the tips of them need to extend into the lumen of the sinusoidal vessels in the marrow in order for the next stage to begin.
• Fragmentation then occurs, as the cytoplasm separating the "beads" on these protoplatelet extensions gets pinched off and the tips of these long processes begin to separate from the rest of the pseudopod, eventually falling away as a mature platelet. Some sort of inhibitory factor is present in the marrow itself which prevents this from taking place until the protoplatelet extensions are dangling in the blood stream.
• This process is "suicidal" and ends with the megakaryocyte exhausting most of its cytoplasm and turning into a wrong-looking cell with a  large multilobular nucleus and minimal other organelles. Apoptosis follows. 
• Thrombopoiesis or megakaryopoiesis are the words used to describe the formation of platelets. This entire process, from commitment to apoptosis, takes 8-10 days.

Regulation of platelet production

• Thrombopoietin,  a huger 332-amino-acid glycoprotein, is probably the most important endocrine stimulator of platelet production. All the relevant progenitor cells (including the bottom-level haematopoietic stem cells) have the receptor for thrombopoietin (c-mpl) and it accelerates the process at each stage of the pathway. Most of it is produced in the liver, and some in the kidney (Sungaran et al, 1997). A recombinant option is available but it probably costs more than your house.
• Regulation of thrombopoietin release is achieved by three main means, described by Kaushansky (2005) in yet another review article :
  • Downregulation is performed by mature platelets themselves, as they seem to have a high-affinity receptor for it. This negative feedback loop makes logical sense: if there are too many platelets, they will scrub the thrombopoietin from the bloodstream, and their production rate will decrease.
  • Upregulation is stimulated by things which, looking at them from an arm's length, you'd be forced to describe as "stress factors" or "inflammatory mediators", for example, IL-6 (which also stimulates the release of CRP).

Life and fate of platelets

They are billions; the average platelet count is 150×10⁹ to 400×10⁹ per liter, which means a healthy bloodstream may have up to three trillion platelets in it. Once in the circulation, a platelet has a lifespan of approximately ten days, which means your bone marrow at a basic steady state is producing enough platelets to increase their count by 15-40×10⁹/L per day. 

Each platelet has an internal apoptotic clock, which runs without a nucleus, and which determines this ten day lifespan (Mason et al, 2007). This clock consists of an antagonistic balance between the pro-survival protein Bcl-xL and pro-apoptosis protein Bak. Bak always wins, and the platelets undergo several changes which include surface phosphatidylserine upregulation, deglycosylation of membrane glycoproteins, and "desialylation" where sialic acid residues disappear from their surface. The upshot is that there is an internal process that gradually moves platelets towards their destruction, and it continues even in storage, which limits the lifespan of refrigerated pooled platelets to about 4-7 days (Ohto et al, 2009). 

Structure of platelets

They are tiny and cute. It would have been lovely to have retained some of the archaic terms for them (in medicine, we have done that for much stupider things), but the author is probably in a minority with his fondness for blutplättchen or poussière de sang ("dust of the blood"). Each platelet is an anuclear self-contained molecular machine packed with the exact things it needs to do its job, including glycogen and mitochondria which it requires for ATP production. This excellent graphic from George (2000) is included here with minimal modification because it represents the important parts very well (but the scan did not work well for the original labels); whereas the best non-pictorial description of platelet structure has got to be Fritzma (2015). 

Not shown is the platelet glycocalyx, which is thicker than most other cellular glycocalyces (20–30 nm), full of necessary procvoagulant molecules and covered in the negative change that repels other platelets.

Function of platelets in the process of haemostasis

To say "they are central to haemostasis" would probably not be enough detail, but would be fairly accurate. Consider any TEG or ROTEM you might have seen of a severely thrombocytopenic patient. To offer a detailed explanation of what platelets actually do would have required a detailed explanation of haemostasis, and that is handled well enough elsewhere. Instead, a brief exam-ready summary will be produced here as a ready answer for questions like Question 4(p.2) from the first paper of 2009.

Role of platelets in primary haemostasis

• Platelets form the initial platelet plug in primary haemostasis, by:
• Adhesion to the denuded surface collagen via VWF, as well as directly
  • Collagen, laminin and other basement membrane components can bind platelets directly
  • These molecules also activate platelets
• Aggregation (platelet to platelet) mediated by fibrin and VWF
  • GPIIb/IIIa receptor protein mediates the linking of platelet to platelet by bridges of fibrinogen, fibrin and VWF
• Activation, which means
  • Degranulation (release of vasoactive and platelet-activating mediators, including ADP, serotonin, Factor V, and various vasoactive eicosanoids including thromboxane A2 which activates yet more platelets)
  • Shape change (flattening and extension of cellular projections)
  • Phosphatidylserine exposure on the platelet surface, which is essential for clotting factor binding
• Vasoconstriction is crucial to primary haemostasis, and this is mainly mediated by the products of platelet degranulation.

Role of platelets in secondary haemostasis

• Activated platelets act as the surface for most of the events which need to take place for secondary haemostasis.
• The exposure of negatively charged phospholipid on the platelet surface is crucial to this
• Amplification
  • Intrinsic pathway activation by the available thrombin and other platelet granule content leads to the increase in available clotting factors in the region of the platelet plug
  • The available thrombin activates factor XI and leads to the activation of FXI
  • Activate platelet surfaces act as sites of attachment for FVIIIa and FVa
• Propagation
  • Platelet-bound Factors FVIIIa  FVa and FX activate thrombin
  • This leads to the formation of a large amount of thrombin (the "thrombin burst")
  • The large amount of thrombin made available allows the generation of a large amount of fibrin from fibrinogen
• Contraction of platelets occurs in later stages of clot maturation